Compositions and methods for modulating dysferlin expression

ABSTRACT

Aspects of the disclosure relate to methods of altering RNA splicing in a subject. In some embodiments, methods are provided for correcting splicing in a cell that contains a DYSF gene having a mutation that results in defective splicing.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of U.S.provisional application U.S. Ser. No. 62/007,397, filed Jun. 3, 2014,and entitled “Compositions and Methods for Modulating DysferlinExpression”, the entire contents of which are incorporated herein byreference.

BACKGROUND

Dysferlin (DYSF) is a large transmembrane protein and a member of theferlin family of Ca²⁺-dependent phospholipid-binding proteins that playa role in membrane vesicle fusion and membrane repair. Insufficientlevels of normal dysferlin lead to specific forms of muscular dystrophy(dysferlinopathies) that include Miyoshi myopathy (MM), limb-girdlemuscular dystrophy type 2B (LGMD-2B) and distal myopathy with anteriortibial onset (DMAT). These recessively-inherited diseases arecharacterized by progressive, muscle weakness with typical onset in lateteens to early twenties, beginning either in proximal (LGMD-2B) or indistal muscles (MM, DMAT) but eventually affecting broader groups ofmuscles similarly. Clinical symptoms include muscle degenerationaccompanied by substantial elevation serum creatine kinase (CK),indicative of muscle damage, inflammation and abnormal musclemorphology. As with other forms of muscular dystrophy, there iscurrently no cure for dysferlinopathies, and a need exists for newtherapeutic approaches to treat these genetic diseases.

SUMMARY

Aspects of the disclosure relate to compositions and methods formodulating dysferlin expression by altering splicing of RNA expressedfrom a DYSF gene. In particular, aspects of the disclosure relate to anew deep intronic mutation identified in dysferlin intron 44i thatalters the splicing of mRNA and leads to the inclusion of a pseudoexonbetween exons 44 and 45 (DYSF PE44.1). DYSF PE44.1 maintains the readingframe, adding 59 amino acids within the conserved C2F domain, anddisrupting function. In some embodiments, an exon-skipping strategy isprovided that induces altered splicing in this PE44.1 mutant region ofDYSF mRNA. In such embodiments, antisense nucleic acids designed toprevent DYSF PE44.1 splicing restore normal mRNA splicing and thusnormal protein (as opposed to a modified or truncated version currentlypossible with other exon targets). Antisense nucleic acids providedherein that target DYSF PE44.1 reduce expression of the mutant mRNAsplice form and restore higher levels of the normal form of mRNA andgreater protein levels.

Aspects of the disclosure relate to a method of modulating splicing in acell that contains a DYSF gene comprising a c.4886+1249 (G>T) mutation.In some embodiments, the method involves delivering to the cell anantisense nucleic acid that targets a pre-messenger RNA expressed fromthe DYSF gene and alters splicing of the pre-messenger RNA such thatexons 44 and 45 of the pre-messenger RNA are spliced together without anintervening pseudoexon. In some embodiments, the cell is heterozygousfor the c.4886+1249 (G>T) point mutation. In some embodiments, the cellis homozygous for the c.4886+1249 (G>T) point mutation. In certainembodiments, the cell contains a second DYSF gene encoding a wild-typeDYSF protein. In some embodiments, the cell contains a second DYSF genecomprising a mutation that causes a premature stop codon. In certainembodiments, the premature stop codon is within a region encoding theC2D domain of DYSF protein. In some embodiments, the second DYSF gene isa human DYSF gene, and the mutation that causes the premature stop codonis a c.3444_3445delTGinsAA mutation. In certain embodiments, the cell isin vitro. In some embodiments, the cell is in vivo. In certainembodiments, the cell is a non-human cell engineered to contain the DYSFgene comprising the c.4886+1249 (G>T) mutation. In some embodiments, thecell is a human cell. In certain embodiments, the human cell isengineered to contain the DYSF gene comprising the c.4886+1249 (G>T)mutation. In some embodiments, the human cell is from a subject having amuscular dystrophy that is associated at least in part with the DYSFgene comprising the c.4886+1249 (G>T) mutation. In certain embodiments,the muscular dystrophy is of the Miyoshi Myopathy-type, or othermuscular dystrophy caused by abnormal expression of the dysferlin geneproduct.

Aspects of the disclosure relate to a method of modulating splicing in acell containing a DYSF gene that encodes a pre-messenger RNA having apseudoexon incorporated between exons 44 and 45. In some embodiments,the method involves delivering to the cell an antisense nucleic acid(e.g., an oligonucleotide) that targets the pre-messenger RNA encoded bythe human DYSF gene and alters splicing of the pre-messenger RNA suchthat the pseudoexon is not incorporated between exons 44 and 45 of thepre-messenger RNA. In some embodiments, the DYSF gene is a human DYSFgene that comprises a c.4886+1249 (G>T) mutation. In certainembodiments, the antisense nucleic acid is an oligonucleotide of 10 to25 nucleotides in length comprising a region of complementarity that iscomplementary with at least 8 contiguous nucleotides of a sequencewithin a region between exons 43 and 46 encoded by the DYSF gene. Insome embodiments, the antisense nucleic acid is an oligonucleotide of 10to 25 nucleotides in length comprising a region of complementarity thatis complementary with at least 8 contiguous nucleotides of a sequencewithin a region between exons 44 and 45 encoded by the DYSF gene. Incertain embodiments, the antisense nucleic acid is an oligonucleotidecomprising a sequence of any one of AON1 to AON20 as set forth in Tables4 and 8. In some embodiments, the antisense nucleic acid is expressedfrom a transgene. In some embodiments, the transgene is delivered to thecell using a viral vector. In certain embodiments, the viral vector is arecombinant AAV vector.

Aspects of the disclosure relate to a method of altering RNA splicing ina subject. In some embodiments, the method involves administering to thesubject an antisense nucleic acid (e.g., an oligonucleotide) thattargets a pre-messenger RNA encoded by a human DYSF gene and alterssplicing of the pre-messenger RNA such that exons 44 and 45 of the RNAare spliced together without an intervening pseudoexon, wherein thehuman DYSF gene comprises a c.4886+1249 (G>T) mutation. In someembodiments, the method further involves, prior to administering theantisense nucleic acid, detecting that the subject has the human DYSFgene that comprises the c.4886+1249 (G>T) mutation. In certainembodiments, the human DYSF gene is detected using a hybridization assaythat discriminates between the presence of a guanosine and a thymidineat position c.4886+1249 of the human DYSF gene. In some embodiments, thehybridization assay is a polymerase chain reaction (PCR) assay. Incertain embodiments, the PCR assay comprises using a primer or probethat is at least partially complementary with a nucleic acid having asequence as set forth in any one of SEQ ID NOs: 117 to 121 or acomplementary sequence thereof. In some embodiments, the antisensenucleic acid is an oligonucleotide of 10 to 25 nucleotides in lengthcomprising a region of complementarity that is complementary with atleast 8 contiguous nucleotides of a sequence within a region betweenexons 43 and 46 encoded by a human DYSF gene. In certain embodiments,the antisense nucleic acid is an oligonucleotide of 10 to 25 nucleotidesin length comprising a region of complementarity that is complementarywith at least 8 contiguous nucleotides of a sequence within a regionbetween exons 44 and 45 encoded by a human DYSF gene. In certainembodiments, the antisense nucleic acid is an oligonucleotide comprisinga sequence of any one of AON1 to AON20 as set forth in Tables 4 and 8.In some embodiments, the antisense nucleic acid is expressed from atransgene. In certain embodiments, the transgene is administered to thesubject using a viral vector. In some embodiments, the viral vector is arecombinant AAV vector.

Aspects of the disclosure relate to oligonucleotides of 10 to 25nucleotides in length comprising a region of complementarity that iscomplementary with at least 8 contiguous nucleotides of a sequencewithin a region between exons 43 and 46 encoded by a human DYSF gene. Insome embodiments, the oligonucleotide comprises at least one modifiednucleotide or at least one modified internucleotide linkage. In someembodiments, the human DYSF gene comprises a c.4886+1249 (G>T) mutation.In certain embodiments, the region of complementarity is complementarywith at least 8 contiguous nucleotides of a sequence as set forth as SEQID NO: 116. In some embodiments, the region of complementarity iscomplementary with at least 8 contiguous nucleotides of a sequence asset forth as SEQ ID NO: 117, 118, 119, 120, or 121. In some embodiments,the region of complementarity is complementary with an exonic spliceenhancer or inhibitor sequence. In certain embodiments, the region ofcomplementarity is complementary with a splice donor motif. In someembodiments, the region of complementarity is complementary with asplice acceptor motif. In certain embodiments, the region ofcomplementarity is complementary with a lariat branch point. In someembodiments, the human DYSF gene comprises a mutation that results anin-frame pseudoexon between exons 44 and 45. In certain embodiments, theoligonucleotide, when present in a cell that contains the human DYSFgene, alters splicing of a pre-messenger RNA expressed from the humanDYSF gene such that the pseudoexon is not incorporated between exons 44and 45. In some embodiments, the oligonucleotide comprises at least onemodified nucleotide. In certain embodiments, the at least one modifiednucleotide is a 2′-modified nucleotide. In some embodiments, the2′-modified nucleotide is a 2′-deoxy, 2′-fluoro, 2′-O-methyl,2′-O-methoxyethyl, 2′-amino and 2′-aminoalkoxy modified nucleotides. Incertain embodiments, the 2′-modified nucleotide comprises a 2′-O-4′-Cmethylene bridge. In some embodiments, the oligonucleotide comprises atleast one modified internucleotide linkage. In certain embodiments, theat least one modified internucleotide linkage is a phosphorothioatemodified linkage. In some embodiments, the oligonucleotide is amorpholino (or modified morpholino, e.g. peptide conjugated morpholino).In certain embodiments, the oligonucleotide comprises alternating LNAand RNA nucleotides. In some embodiments, the oligonucleotide comprisesalternating LNA and DNA nucleotides. In certain embodiments, theoligonucleotide comprises alternating RNA and DNA nucleotides. In someembodiments, the oligonucleotide, when present in a cell that containsthe human DYSF gene, is capable of hybridizing with RNA expressed fromthe human DYSF gene without inducing cleavage of the RNA by an RNase. Incertain embodiments, the oligonucleotide comprises a sequence of any oneof AON1 to AON20 as set forth in Tables 4 and 8. In some embodiments,the oligonucleotide comprises a region of complementarity that iscomplementary with at least 8 contiguous nucleotides of a sequencewithin a region between exons 44 and 45 encoded by a human DYSF gene.Further aspects of the disclosure relate to compositions comprising oneor more oligonucleotides disclosed herein and a carrier. In someembodiments, the composition is a pharmaceutical composition comprisinga pharmaceutically-acceptable carrier.

Aspects of the disclosure relate to a preparation of oligonucleotides,in which at least 95% (e.g., at least 98%, at least 99%) of theoligonucleotides are 10 to 25 nucleotides in length and comprise aregion of complementarity that is complementary with at least 8contiguous nucleotides of a sequence within a region between exons 43and 46 encoded by a human DYSF gene. In some embodiments, the region ofcomplementarity is complementary with at least 8 contiguous nucleotidesof a sequence as set forth in SEQ ID NO: 116. In certain embodiments,the region of complementarity is complementary with at least 8contiguous nucleotides of a sequence as set forth in SEQ ID NO: 117,118, 119, 120 or 121. In some embodiments, the region of complementarityis complementary with an exonic splice enhancer or inhibitor sequence.In certain embodiments, the region of complementarity is complementarywith a splice donor motif. In some embodiments, the region ofcomplementarity is complementary with a splice acceptor motif. Incertain embodiments, the region of complementarity is complementary witha lariat branch point. In some embodiments, the human DYSF genecomprises a mutation that results an in-frame pseudoexon between exons44 and 45. In certain embodiments, the oligonucleotide, when present ina cell that contains the human DYSF gene, alters splicing of an RNAexpressed from the human DYSF gene such that the pseudoexon is notincorporated between exons 44 and 45. In some embodiments, theoligonucleotides are lyophilized. In certain embodiments, at least 95%,at least 98%, or at least 99% of the oligonucleotides are identical. Insome embodiments, the oligonucleotides comprises a sequence of any oneof AON1 to AON20 as set forth in Tables 4 and 8. In certain embodiments,the preparation comprise a region of complementarity that iscomplementary with at least 8 contiguous nucleotides of a sequencewithin a region between exons 44 and 45 encoded by a human DYSF gene.

Aspects of the disclosure relate to kits comprising a container housinga preparation disclosed herein.

Aspects of the disclosure relate to a pair of PCR primers havingsequences selected from the primer sequences set forth Table 1 or Table5.

Aspects of the disclosure relate to compositions comprising a pair ofPCR primers disclosed herein and a template comprising at least aportion of a human DYSF gene.

Further aspects of the disclosure relate to a composition comprising apair of PCR primers disclosed herein and a probe having as sequence asset forth in Table 3.

Still further aspects of the disclosure relate to a labelledoligonucleotide comprising a sequence set forth as: CACCTACCACTTCCCTCCA(SEQ ID NO: 51) or ACCTACCACTTACCTCCA (SEQ ID NO: 52) or a fragmentthereof that is useful for detecting presence or absence of ac.4886+1249 (G>T) mutation in a DYSF gene. In certain aspects, thedisclosure relates to an expression construct encoding an antisensenucleic acid having a region of complementarity that is complementarywith a sequence between exons 43 and 46 encoded by a human DYSF gene. Insome embodiments, the human DYSF gene comprises a c.4886+1249 (G>T)mutation. In certain embodiments, the region of complementarity iscomplementary with at least 8 contiguous nucleotides of a sequence asset forth as SEQ ID NO: 116. In some embodiments, the region ofcomplementarity is complementary with at least 8 contiguous nucleotidesof a sequence as set forth as SEQ ID NO: 117, 118,119, 120 or 121. Incertain embodiments, the region of complementarity comprises a sequencecomplementary with an exonic splice enhancer or inhibitor sequence. Insome embodiments, the region of complementarity comprises a sequencecomplementary with a splice donor motif. In certain embodiments, theregion of complementarity comprises a sequence complementary with asplice acceptor motif. In some embodiments, the region ofcomplementarity comprises a sequence complementary with a lariat branchpoint. In certain embodiments, the antisense nucleic acid expressed fromthe vector, when present in a cell that contains the human DYSF gene,alters splicing of a pre-messenger RNA expressed from the human DYSFgene such that the pseudoexon is not incorporated between exons 44 and45.

Further aspects of the disclosure relate to a recombinant AAV or otherviral vector comprising an expression construct disclosed herein.

According to some aspects of the disclosure, an engineered cell isprovided that comprises an exogenous human DYSF gene having ac.4886+1249 (G>T) mutation, in which the cell expresses a pre-messengerRNA from the exogenous DYSF gene containing a pseudoexon between exons44 and 45. In some embodiments, the cell is not of a human origin. Incertain embodiments, the cell is of a human origin. In some aspects, acomposition is provided that comprises cells containing a human DYSFgene having a c.4886+1249 (G>T) mutation and an artificial cell culturemedium. In some embodiments, a tissue culture system is provided thatcomprises such a composition. In some embodiments, the compositionfurther comprise an oligonucleotide disclosed herein, e.g., whicholigonucleotide has been delivered to the cells to modulate DYSFexpression.

In still further aspects of the disclosure a transgenic organism isprovided that comprises an engineered cell disclosed herein. In someembodiments, the organism is a mammal. In some embodiments, the mammalis a rodent. In some embodiments, the rodent is mouse or rat.

In some aspects, the disclosure provides an antibody or antigen bindingfragment thereof that binds selectively to a DYSF variant or portionthereof. In some embodiments, the DYSF variant or portion thereofcomprises or consists of a peptide encoded by SEQ ID NO: 116. In someembodiments, the antibody targets the C-terminal portion of the peptideencoded by SEQ ID NO: 116. In some embodiments, the antibody targets anamino acid sequence as set forth in SEQ ID NO: 122.

In some aspects, the disclosure provides a method of identifying and/ortreating a DYSF intronic mutation, the method comprising: (a) amplifyingand/or sequencing a nucleic acid obtained from or prepared from abiological sample obtained from a subject (e.g., using at least oneprimer set as set forth in Table 1, Table 2 and/or Table 3) to detectthe presence of a c.4886+1249 (G>T) mutation in a DYSF gene; or (b)performing an immunoassay (e.g., an ELISA, Western blot, e.g., using ananti-PE44.1 antibody) on a biological sample obtained from a subject;and, (c) diagnosing or aiding in diagnosing the subject has having anintronic mutation in the DYSF gene based upon detection of thec.4886+1249 (G>T) mutation in (a) or detection of the protein productencoded by PE44.1 in (b).

In some aspects, the disclosure provides a method of identifying and/ortreating a DYSF intronic mutation, the method comprising: (a) amplifyingthe cDNA of a biological sample obtained from a subject using at leastone primer set as set forth in Table 1, Table 2 and/or Table 3 to detectthe presence of a c.4886+1249 (G>T) mutation in a DYSF gene; or (b)performing a Western blot on a biological sample obtained from asubject, wherein the Western blot is probed with an anti-PE44.1antibody; and, (c) diagnosing the subject has having an intronicmutation in the DYSF gene based upon detection of the c.4886+1249 (G>T)mutation in (a) or detection of the protein product encoded by PE44.1 in(b).

In some embodiments, the biological sample is blood or muscle tissue. Insome embodiments, the blood comprises monocytes. In some embodiments,the monocytes are isolated, optionally by cell a sorting method.

In some embodiments, the method further comprises administering to thesubject an antisense oligonucleotide (e.g., an antisense oligonucleotideas described by the disclosure).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. (A) Myosin Heavy Chain (MHC) expression in iFDMS derived frompatient P1 dermal fibroblasts stained with anti-MHC antibody. Nuclei arestained blue. (Scale bar 100 lam). (B) Western blots show dysferlinprotein in iFDMs from patients P1 and P2, normal fibroblasts (N), andhuman UBic myotubes (Mt). iFDMs from patients express lower levels ofDYSF than normal iFDMs, and this appears to be of normal size. GAPDHlevels are shown as a control for protein loading.

FIG. 2. (A) Identification of the DYSF intron 44i mutation. RT-PCR ofcDNA from two patients (P1, P2) shows novel amplicons in patients(arrows) generated using with two primer sets (panels B, C) but not withany other sets (panels A, D), indicating the presence of additionalsequence within the patient cDNA in the region amplified by primer setsused in panels B and C. The product was sequenced to identify theinserted sequence. (B) RT-PCR of cDNA from skeletal muscle tissue frompatient P1 shows very low-level expression of DYSF mRNA compared tomuscle from a normal control individual (N), and this includes mutant(320 bp) and wild-type (143 bp) transcripts for this locus detected byprimers that flank the exon 44/45 junction. The top panel is a brightergel exposure than the bottom two panels, allowing visualization of thePCR products in patient muscle. Lower two panels show the same exposurefor both the DYSF and B2M loading control PCR products (123 bp).

FIG. 3. cDNA sequencing shows that patients P1 and P2 have an insertionof insertion 177 nt at the junction of exons 44 and 45. This insertionmaintains the reading frame and leads to inclusion of 59 additionalamino acids in the protein sequence. The mRNA from these patientstherefore contains a novel pseudoexon, PE44.1, spliced into the codingsequence.

FIG. 4. A novel deep intronic point mutation leads to inclusion ofPE44.1 in the mature mRNA of patients P1 and P2. (A) Genomic sequencingof DYSF intron 44i shows patients are heterozygous for a point mutation(c.4886+1249 (G>T)). The location of this 44i mutation, indicated withan asterisk (*), creates a novel splice donor consensus sequence at the3′ end of the PE44.1 sequence (B). (C) The intronic sequence upstream ofPE44.1 contains additional consensus sites required for mRNA splicingincluding a splice acceptor site at the 5′ end of PE44.1, an adjacentpyrimidine-rich region and two potential lariat branch point consensussequences that could be used to promote splicing. These sequences, inthe presence of (c.4886+1249 (G>T)) mutation, allow the new pseudoexonPE44.1 to be spliced between exons 44 and 45.

FIG. 5. Normal and mutant PE44.1 mRNA are expressed through alternativesplicing of the DYSF transcripts. (A) The mRNA structure within the DYSFexon 44-intron 44i -exon 45 region is shown along with the normal andmutant splicing patterns. Numbers along the

RNA indicate the size of each element in bp. The site of the 44imutation (c.4886+1249 (G>T)) is indicated with an asterisk (*). (B, C)The normal and mutant splice product (including PE44.1) are shown(displaying the region of DYSF from exons 38-51). Also shown is thenormal and mutant protein. PE44.1 results in a 59 amino acids insertionwithin the normal C2F domain (normally 84 aa), creating a largedisruption in this region.

FIG. 6. Analysis of genomic DNA from four immediate relatives ofpatients P1 and P2 reveal the inheritance pattern of the two mutationsin this family. (A) Genomic DNA from each family member (listed byglobal I.D.) was amplified and sequenced using primers flanking thepreviously known DYSF exon 32 mutation in these patients as well as thenew DYSF 44i mutation associated with PE44.1 expression. Mutantnucleotides are shown in red. (B) Results show maternal inheritance ofthe DYSF 44i mutation.

FIG. 7. Antisense oligonucleotide-mediated skipping of PE44.1 in iFDMcells from patients P1 and P2. (A) AON1, AON2 and AON3 (see Table 4)target potential exonic splicing enhancers in PE44.1 in the areas shown.Primers within exon 44 and 45 were used to amplify cDNAs from iFDMstreated with AONs to distinguish normal mRNA transcripts (143 bp productcontaining exon 44+45) from mutant PE44.1 transcripts (320 bp productcontaining exon 44 +PE44.1+45). (B) iFDM cells from Patient P1 treatedwith AON2 and AON3 (duplicate cultures for each) expressed reducedamounts of PE44.1 mutant mRNA and slightly higher normal DYSF mRNAcompared with AON1-treated, non-specific scrambled (SCR) AON-treated orTE treated cells, which showed approximately equal proportions of mutantPE44.1 and normal mRNAs. (C) A separate experiment using patient P1, P2and normal iFDMs (duplicate cultures for each treatment) showed thatAON2 and AON3 treatments again reduced PE44.1 mutant mRNA expression andincreased the relative abundance of normal DYSF mRNA. As expected,normal iFDMs only expressed normal DYSF transcripts. (D) QuantitativeRT-PCR analysis of the same RNAs in panel C show that treatment ifpatient iFDMs with AON2 and AON3 significantly reduces the expression ofthe mutant transcripts (gray bars, mean ±SD, *p<0.05) and increasesexpression of the normal transcripts (black bars, mean ±SD, **, p<<0.05)compared to SCR and TE controls. The relative expression of each form iscalculated using the amplification of a PCR product spanning the exon50/51 junction as representative of 100% DYSF expression.

FIG. 8. Treatment of patient iFDMs with AONS directed to PE44.1 inducesdysferlin protein expression. TMX-induced iFDMs (duplicate or triplicatecultures as shown) were cultured in DM for 6 days then treated with AONsin DM (or TE buffer or medium only (0) as controls). Cells werecollected for protein analysis 1, 3 and 8 days after AON addition. Forthe 8-day samples, medium with AONS was removed on day 3 and replacedwith DM only. (A) Western blots show that after 1 day of AON treatmentthere are no changes in DYSF protein expression, but DYSF levels wereincreased in AON3-treated cultures 3 and 8 days after AON addition.GAPDH expression served as a control for protein loading (5 μgprotein/lane). (B) Quantitation of the bands shown in panel A revealsthat there is a significant increase in DYSF protein in AON3-treatedcultures compared with SCR oligo-treated controls (mean ±SD, *p<0.05).Protein levels in normal control iFDM cultures are not affected by AONtreatments.

FIG. 9. AON3 treatment of patient iFDMs alters expression of DYSF PE44.1mutant and normal mRNA splice forms at 1, 3 and 8 days after AONaddition. (A) RT-PCR analysis of mRNA from cultures collected inparallel with those in FIG. 8 show that treatment if Patient 1 (P1) andPatient 2 (P2) iFDMs with AON3 reduces the expression of the PE44.1mutant transcript and increases normal transcript expression. NormaliFDMs (N) express only the normal DYSF form, not affected by AONtreatments. (B) Quantitative RT-PCR analysis of the same mRNAs confirmsthe effects of AON3 treatment. (PE44.1 mutant transcript expression:gray bars; normal transcript expression: black bars). The relativeexpression of each form is calculated using exon 50/51 amplicon levelsas 100% DYSF expression as in FIG. 7.

FIG. 10. Anti-PE44.1 antibody recognizes a protein (˜237 kDa) in patientP1 and P2 iFDMs but not in normal (N) iFDMs. Protein blots (125 pg/lane)were probed with an antibody against PE44.1 or normal DYSF (Hamlet). P1and P2 show reduced levels of normal DYSF compared with normal cells.Lower panels, Ponceau S stain of total proteins bound to the blot showseven protein loading.

DETAILED DESCRIPTION

Aspects of the disclosure relate to methods of altering RNA splicing ina subject. In some embodiments, the disclosure relates to compositionsand methods for modulating splicing in a cell that contains a DYSF genehaving a mutation that results an in-frame pseudoexon between exons 44and 45 that results in defects in dysferlin protein expression. Thereare a number of specific features of the dysferlin protein thatcontribute to its function and interaction with other proteins. As amember of the ferlin family, dysferlin has seven Ca²⁺-sensitivephospholipid binding C2 domains (C2A through C2G) that vary in theirphospholipid binding characteristics, relative importance for dysferlindimerization and membrane interaction but collectively may play a rolein altering the structure or curvature of lipid bilayers facilitatingmembrane fusion and interaction with other membrane associated proteins.Dysferlin interacts with a number of proteins that function in membranetrafficking and fusion including caveolin-3, annexins, affixin,calpain-3 and AHNAK. Through these interactions, dysferlin plays animportant role in sarcolemma repair following membrane damage, as wellas vesicle trafficking, membrane turnover, and T-tubule formation andstability.

Synthesis of the large (237 kDa) dysferlin is a protein is involved. Theprotein is derived from a ˜6.2 kb transcript assembled from up to 55exons. There are 14 isoforms of DYSF that can arise from use of twoseparate promoters and alternate exon splicing, with isoform 8 being thepredominant form in skeletal muscle. Disease-causing mutations indysferlin occur throughout the gene. Genetic data compiled in UniversalMutation Database for Dysferlin (UMD-DYSF, v.1.1 April 26,2013) lists337 disease-causing mutations that have been found in 725 patientsworldwide. Approximately 48% of these patients are homozygous forspecific mutations and approximately 52% are heterozygous. Roughly 17%of patients have only one mutant allele identified, and it is likelythat the unknown allele carries a mutation within intronic or regulatoryregions that are not identifiable through standard exome sequencingapproaches. Alternative approaches are required to identify these othermutant alleles.

In some embodiments, there are at least 14 variants (e.g., isoforms) ofDYSF transcripts that arise from the use of two separate promotersand/or alternate exon splicing events. In some embodiments, variant 8 isa predominant form of DYSF in skeletal muscle. In some embodiments, DYSFmRNA variant 8 (NM_003494.3) is used as a reference sequence for RNA andcDNA analyses and/or as a context for specifying exon and intron numericassignments and nomenclature.

Some aspects of the present disclosure relate to the identification of anovel mutant allele in certain Miyoshi myopathy (MM) patients that haveonly one of their mutant alleles identified. In some embodiments, suchpatients lack normal dysferlin in their muscles and are heterozygous fora nonsense mutation in an exon of one allele of the DYSF gene but theother disease causing mutation has not been identified despite multipleexome sequencing efforts. Some aspects of the present disclosure relateto the identification of a deep intronic point mutation within intron 44(44i) that leads to abnormal mRNA and protein structure. Other aspectsof the present disclosure relate to the partial restoration of normalDYSF mRNA and protein levels in myogenic cells from these patients usingantisense nucleic acids to by-pass the effects of the 44i mutation,providing a novel therapeutic strategy to restore DYSF function.

Accordingly, methods are provided herein that involve delivering to thecell an antisense nucleic acid that targets a pre-messenger RNAexpressed from the DYSF gene and alters splicing of the pre-messengerRNA such that exons 44 and 45 of the pre-messenger RNA are splicedtogether without an intervening pseudoexon. In some embodiments, theDYSF gene comprises a c.4886+1249 (G>T) mutation. In this context,c.4886+1249 refers to the mutational position relative to the ATG startcodon of the transcript (the coding sequence) for dysferlin using thepredominant mRNA in muscle, isoform 8 (NCBI Reference Sequence:NM_003494.3), as the reference. c.4886 is the last nucleotide of exon44. The mutation is at the1249th nucleotide after this position, withinthe intron (44i). Normally this 1249th nucleotide is a “G”, but certaindiseased patients this is mutated to a “T”. In some embodiments, thecell is heterozygous for the c.4886+1249 (G>T) point mutation.

As used herein, the term, “antisense nucleic acid,” refers to a nucleicacid that has sequence complementarity to a target sequence and isspecifically hybridizable, e.g., under stringent conditions, with anucleic acid having the target sequence. An antisense nucleic acid isspecifically hybridizable when binding of the antisense nucleic acid tothe target nucleic acid is sufficient to produce complementary basedpairing between the antisense nucleic acid and the target nucleic acid,and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense nucleic acid to non-target nucleicacid under conditions in which specific binding is desired, e.g., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed. In some embodiments, an antisense nucleic acidis used that has a region of complementarity that is perfectlycomplementary to a portion of a target nucleic acid (e.g., target RNA).However, it should be appreciated that in some embodiments, an antisensenucleic acid may be used that has less than 100% sequencecomplementarity with a target nucleic acid. An antisense nucleic acidoligonucleotide may comprise a region of complementarity that iscomplementary with sequence as set forth in SEQ ID NO: 116, 117, 118 or119. The region of complementarity of the antisense nucleic acid may becomplementary with at least 6, e.g., at least 7, at least 8, at least 9,at least 10, at least 15 or more consecutive nucleotides of a targetnucleic acid. In addition, to minimize the likelihood of off-targeteffects, an antisense nucleic acid may be designed to ensure that itdoes not have a sequence (e.g., of 5 or more consecutive nucleotides)that is complementary with an off-target nucleic acid.

Complementary refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anantisense nucleic acid is capable of hydrogen bonding with a nucleotideat the corresponding position of a target nucleic acid (e.g., targetRNA), then the antisense nucleic acid and target nucleic acid areconsidered to be complementary to each other at that position. Theantisense nucleic acid and target nucleic acid are complementary to eachother when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides that can hydrogen bond with eachother through their bases. Thus, “complementary” is a term that is usedto indicate a sufficient degree of complementarity or precise pairingsuch that stable and specific binding occurs between the antisensenucleic acid and target nucleic acid. However, it should be appreciatedthat 100% complementarity is not required. For example, in someembodiments, an antisense nucleic acid (e.g., an oligonucleotide) may beat least 80% complementary to (e.g., at least 85%, 90%, 91%, 92%, 93%,940%, 95%, 96%, 97%, 98%, 99% or 100% complementary to) the consecutivenucleotides of a target nucleic acid.

Thus, it is understood in the art that a complementary nucleotidesequence need not be 100% complementary to that of its target to bespecifically hybridizable. In some embodiments, a complementary nucleicacid sequence for purposes of the present disclosure is specificallyhybridizable when binding of the sequence to the target nucleic acidproduces the desired alterations in splicing to occur and there is asufficient degree of complementarity to avoid non-specific binding tonon-target nucleic acids under conditions in which avoidance ofnon-specific binding is desired, e.g., under physiological conditions inthe case of in vivo assays or therapeutic treatment, and in the case ofin vitro assays, under conditions in which the assays are performedunder suitable conditions of stringency.

Sequence identity, including determination of sequence complementarityfor nucleic acid sequences, may be determined by sequence comparison andalignment algorithms known in the art. To determine the percent identityof two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the first sequenceor second sequence for optimal alignment). The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same residue as the correspondingposition in the second sequence, then the molecules are identical atthat position. In some embodiments, the percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (e.g., % homology=# of identical positions/total # ofpositions×100), optionally penalizing the score for the number of gapsintroduced and/or length of gaps introduced.

In some embodiments, an antisense nucleic acid is an antisenseoligonucleotide (AON), which may be referred to simply as anoligonucleotide. For example, in some embodiments, oligonucleotides areprovided that comprise a region of complementarity that is complementarywith at least 8, at least 9, at least 10, at least 11, at least 12, atleast 13, at least 14, at least 15 or more contiguous nucleotides of asequence within a region between exons 44 and 45 encoded by a human DYSFgene. Such oligonucleotides are useful for modulating splicing ofdysferlin and prevent incorporation of a pseudoexon between exons 44 and45. In some embodiments, an antisense nucleic acid is an antisenseoligonucleotide (AON) recited in Table 4 or Table 8.

In some embodiments, oligonucleotides of the disclosure have a length ina range of 5 to 40 nucleotides, 5 to 30 nucleotides, 10 to 30nucleotides, 10 to 25 nucleotides, or 15 to 25 nucleotides. In someembodiments of the disclosure, oligonucleotides have a length of 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more. Insome embodiments, the oligonucleotide comprises a region ofcomplementarity that is complementary with a region within 5, 10, 15,25, 50, 100 or 200 nucleotides of a c.4886+1249 (G>T) mutation in ahuman DYSF gene. In some embodiments, the oligonucleotide comprises aregion of complementarity that is complementary with at least 8contiguous nucleotides of a sequence as set forth as SEQ ID NO: 116. Insome embodiments, the oligonucleotide comprises a region ofcomplementarity that is complementary with at least 8 contiguousnucleotides of a sequence as set forth as SEQ ID NO: 117, 118 or 119.The region of complementarity may be complementary with an exonic spliceenhancer or inhibitor sequence, a splice donor motif, a splice acceptormotif or a lariat branch point encoded by a human DYSF gene (e.g.,within a region spanning from exon 44 to exon 45).

In some embodiments, antisense nucleic acids (e.g., oligonucleotides)are provided in a homogeneous preparation, e.g., in which at least 85%,at least 90%, at least 95%, or at least 99% of the oligonucleotides areidentical. For example, in some embodiments, homogeneous preparations ofoligonucleotides are provided in which at least 85%. at least 90%. atleast 95%. or at least 99% of the oligonucleotides in the preparationare 10 to 25 nucleotides in length and comprise a region ofcomplementarity that is complementary with at least 8 contiguousnucleotides of a sequence within a region between exons 43 and 46encoded by a human DYSF gene.

Antisense nucleic acids of the disclosure may be modified to achieve oneor more desired properties, such as, for example, improved cellularuptake, improved stability, reduced immunogenicity, improved potency,improved target hybridization, susceptibility to RNAse cleavage, etc. Insome embodiments, an antisense nucleic acid is modified such that whenpresent in a cell that contains a human DYSF gene, it is capable ofhybridizing with RNA expressed from the human DYSF gene without inducingcleavage of the RNA by an RNase. Antisense nucleic acids can be modifiedat a base moiety, sugar moiety and/or phosphate backbone. Accordingly,antisense nucleic acids may have one or more modified nucleotides (e.g.,a nucleotide analog) and/or one or more backbone modifications (e.g., amodified internucleotide linkage). Antisense nucleic acids may have acombination of modified and unmodified nucleotides. Antisense nucleicacids may also have a combination of modified and unmodifiedinternucleotide linkages.

Antisense nucleic acids may include ribonucleotides,deoxyribonucleotides, and combinations thereof. Examples of modifiednucleotides which can be used in antisense nucleic acids include, forexample, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil. 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, and2,6-diaminopurine.

In some embodiments, a modified nucleotide is a 2′-modified nucleotide.For example . the 2′-modified nucleotide may be a 2′-deoxy, 2′-fluoro,2′-O-methyl, 2′-O-methoxyethyl, 2′-amino and 2′-aminoalkoxy modifiednucleotides. In some embodiments, the 2′-modified nucleotide comprises a2′-O-4′-C methylene bridge, such as a locked nucleic acid (LNA)nucleotide. In some embodiments of a 2′ modified nucleotide the2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugarring thereby forming a bicyclic sugar moiety. In such embodiments, thelinkage may be a methelyne (—CH2—)_(n) group bridging the 2′ oxygen atomand the 3′ or 4′ carbon atom wherein n is 1 or 2.

Antisense nucleic acids may include combinations of LNA nucleotides andunmodified nucleotides. Antisense nucleic acids may include combinationsLNA and RNA nucleotides. Antisense nucleic acids may includecombinations LNA and DNA nucleotides. A further preferredoligonucleotide modification includes Locked Nucleic Acids (LNAs) inwhich the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of thesugar ring thereby forming a bicyclic sugar moiety.

Antisense nucleotide acids may also include nucleobase-modifiednucleotides, e.g., nucleotides containing a non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase, for example.Examples of modified nucleobases include, but are not limited to,uridine and/or cytidine modified at the 5-position, e.g.,5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and/or guanosinesmodified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides,e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyladenosine are suitable. It should be noted that the above modificationsmay be combined.

Within antisense nucleic acids (e.g., oligonucleotides) of thedisclosures, as few as one and as many as all nucleotides can bemodified. For example, an oligonucleotide (e.g., an oligonucleotide of20 nucleotides in length) may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 modified nucleotides. In someembodiments, a modified oligonucleotide will contain as few modifiednucleotides as are necessary to achieve a desired level of in vivostability and/or bioaccessibility or other desired property.

Certain antisense nucleic acids may include nonionic DNA analogs, suchas alkyl- and aryl-phosphates (in which the charged phosphonate oxygenis replaced by an alkyl or aryl group), phosphodiester andalkylphosphotriesters, in which the charged oxygen moiety is alkylated.Nucleic acids which contain a diol, such as tetraethyleneglycol orhexaethyleneglycol, at either or both termini have also been shown to besubstantially resistant to nuclease degradation and may be used herein.In some embodiments, antisense nucleic acids may include at least onelipophilic substituted nucleotide analog and/or a pyrimidine-purinedinucleotide.

In some embodiments, antisense nucleic acids (e.g., oligonucleotides)may have one or two accessible 5′ ends. It is possible to createmodified oligonucleotides having two such 5′ ends, for instance, byattaching two oligonucleotides through a 3′-3′ linkage to generate anoligonucleotide having one or two accessible 5′ ends. The 3′3′-linkagemay be a phosphodiester, phosphorothioate or any other modifiedinternucleoside bridge. Additionally, 3′3′-linked oligonucleotides wherethe linkage between the 3′ terminal nucleosides is not a phosphodiester,phosphorothioate or other modified bridge, can be prepared using anadditional spacer, such as tri- or tetra-ethylenglycol phosphate moiety.

A phosphodiester internucleotide linkage of an antisense nucleic acidcan be replaced with a modified linkage. The modified linkage may beselected from, for example, phosphorothioate, phosphorodithioate,NR1R2-phosphoramidate, boranophosphate, α-hydroxybenzyl phosphonate,phosphate-(C1-C21)-O-alkyl ester,phosphate-[(C6-C12)aryl-(C1-C21)-O-alkyl]ester, (C1-C8)alkylphosphonateand/or (C6-C12)arylphosphonate bridges, and(C7-C12)-α-hydroxymethyl-aryl.

A phosphate backbone of the antisense nucleic acid can be modified togenerate peptide nucleic acid molecules. As used herein, the terms“peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g.,DNA mimics, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols, for example.

Antisense nucleic acids can also be formulated as morpholinooligonucleotides. In such embodiments, the riboside moiety of eachsubunit of an oligonucleotide of the oligonucleotide reagent isconverted to a morpholine moiety. Morpholinos may also be modified, e.g.as peptide conjugated morpholino)

In other embodiments, the antisense nucleic acid (e.g., oligonucleotide)can be linked to functional groups such as peptides (e.g., for targetinghost cell receptors in vivo), or agents facilitating transport acrossthe cell membrane or the blood-brain barrier. Oligonucleotide reagentsof the disclosure also may be modified with chemical moieties (e.g.,cholesterol) that improve the in vivo pharmacological properties of theoligonucleotide reagents.

Treatment Methods

Aspects of the disclosure relate to methods of altering RNA splicing ina subject involving administering to the subject an antisense nucleicacid that targets a pre-messenger RNA encoded by a human DYSF gene andalters splicing of the pre-messenger RNA such that exons 44 and 45 ofthe RNA are spliced together without an intervening pseudoexon. In someembodiments, the subject is heterozygous a c.4886+1249 (G>T) mutation inthe DYSF gene. However, in some embodiments, the subject is homozygous ac.4886+1249 (G>T) mutation in the DYSF gene. Often the subject has or issuspected of having muscular dystrophy (e.g., of the MiyoshiMyopathy-type) caused by abnormal expression of the dysferlin geneproduct and the methods are being implemented for purposes of treatingthe muscular dystrophy. Treating, in this case, includes improvingdysferlin expression and/or ameliorating one or more symptoms ofmuscular dystrophy.

Any appropriate antisense nucleic acid disclosed herein may beadministered. For example, the antisense nucleic acid may be anoligonucleotide (e.g., of 10 to 25 nucleotides in length) comprising aregion of complementarity that is complementary with a sequence within aregion between exons 43 and 46 encoded by a human DYSF gene. Anantisense oligonucleotide may for example comprise a sequence of AON1 toAON20 as set forth in Tables 4 and 8.

In some embodiments, an antisense nucleic acid is expressed from atransgene, e.g., as an antisense RNA transcript. A transgene may beadministered to a subject in a DNA expression construct that isengineered to express an antisense RNA transcript in a subject. A DNAexpression construct may be administered directly or using a viralvector (e.g., a recombinant AAV (rAAV) vector) or other suitable vector.Viral vectors that have been used for gene therapy protocols include,but are not limited to, retroviruses, other RNA viruses such aspoliovirus or Sindbis virus, adenovirus, adeno-associated virus (AAV),herpes viruses, SV 40, vaccinia, lentivirus and other DNA viruses.

Alternatively, a transgene may be express ex vivo and the resultingantisense RNA transcript may be administered directly to the subject.

As disclosed herein antisense nucleic acids (including DNA expressionconstructs that may be used to expressed them) may be administered byany suitable route. For use in therapy, an effective amount of theantisense nucleic acid (e.g. oligonucleotide) and/or other therapeuticagent can be administered to a subject by any mode that delivers theagent to the desired tissue, e.g., muscle tissue. In some embodiments,agents (e.g., antisense nucleic acids) are administered intramuscularly.Other suitable routes of administration include but are not limited tooral, parenteral, intravenous, intraperitoneal, intranasal, sublingual,intratracheal, inhalation, subcutaneous, ocular, vaginal, and rectal.Systemic routes include oral and parenteral. Several types of devicesare regularly used for administration by inhalation. These types ofdevices include metered dose inhalers (MDI), breath-actuated MDI, drypowder inhaler (DPI), spacer/holding chambers in combination with MDI,and nebulizers.

For oral administration, the agents can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the agents of thedisclosure to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Pharmaceutical preparations fororal use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

Pharmaceutical preparations that can be used orally include push fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active agents may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used.

Such microspheres have been well defined in the art. Formulations fororal administration are typically in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner. For administration byinhalation, agents (e.g., antisense nucleic acids) for use according tothe present disclosure may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The agents (e.g., antisense nucleic acids), when it is desirable todeliver them systemically, may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of agents (e.g., antisense nucleic acids) inwater-soluble form. Additionally, suspensions of agents may be preparedas appropriate oily injection suspensions. Suitable lipophilic solventsor vehicles include fatty oils such as sesame oil, or synthetic fattyacid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances that increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents that increase the solubility of theagents to allow for the preparation of highly concentrated solutions.Alternatively, agents (e.g., antisense nucleic acids) may be in powderform for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use. Agents (e.g., antisense nucleic acids)may also be formulated in rectal or vaginal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the agents (e.g., antisense nucleic acids),increasing convenience to the subject and the physician. Many types ofrelease delivery systems are available. They include polymer basesystems such as poly(lactide glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Delivery systems also include non-polymersystems that are: lipids including sterols such as cholesterol,cholesterol esters and fatty acids or neutral fats such as mono , di ,and tri glycerides; hydrogel release systems; silastic systems;peptide-based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; andothers disclosed herein.

Analytical Methods

In some aspects, the disclosure relates to the methods for detectingpresence of a mutation causing an insertion in between exon 44 and exon45 of the DYSF gene. In some embodiments, a clinical sample is obtainedfrom a subject for the purpose of identifying a mutation. As usedherein, a clinical sample refers to a specimen of biological matterobtained from a subject for the purpose of analysis or diagnosis.Non-limiting examples of clinical samples include blood, saliva, urine,feces, tissue, semen, cerebral spinal fluid, nucleic acids, epithelialcells, sweat, tears, hair and mucous.

In some embodiments, a clinical sample may be obtained from the blood ofthe patient. In some embodiments, a clinical sample may be obtained fromthe cells in the blood of a subject. In some embodiments, the cells maybe blood cells. In some embodiments, the cells may be monocytes. In someembodiments, a clinical sample may be obtained from the tissue of asubject. In some embodiments, the tissue of the subject is muscletissue. In some embodiments, the muscle tissue comprises skeletalmuscle. In some embodiments, the muscle tissue comprises smooth muscle.In some embodiments, the muscle tissue comprises cardiac tissue. In someembodiments the clinical sample is obtained from a tissue that is not amuscle tissue. In some embodiments the non-muscle tissue comprisesmesenchymal cells. In some embodiments, the tissue of the subject isskin tissue. In some embodiments, the non-muscle tissue comprisesfibroblasts. In some embodiments, a non-muscle tissue comprises stemcells, including, for example, embryonic stem cells, tissue stem cells,umbilical cord stem cells, mesenchymal stem cells, induced pluripotentstem cells, multipotent stem cells, totipotent stem cells, unipotentstem cells, progenitor cells, blastocysts, bone marrow stromal cells,hematopoietic stem cells, oligopotent stem cells, neural stem cells, andtrophoblast stem cells. In some embodiments, methods may involvegenotyping a subject with respect to the human DYSF gene for purposes ofselecting an appropriate treatment for the subject. For example, asubject may be administered an antisense nucleic acid disclosed hereinif it is determined that the subject has a DYSF gene having a mutationthat results an in-frame pseudoexon being coded for between exons 44 and45 (e.g., a c.4886+1249 (G>T) mutation.) Often the subject has or issuspected of having muscular dystrophy (e.g., of the MiyoshiMyopathy-type) caused by abnormal expression of the dysferlin geneproduct.

The genotype of the subject may be assessed using a hybridization assaythat discriminates between the presence of a guanosine and a thymidineat position c.4886+1249 of the human DYSF gene. An example of a suitablehybridization is a polymerase chain reaction (PCR) based allelicdiscrimination assay. A PCR based assay may be performed, for example,by using a primers that are at least partially complementary with anucleic acid having a sequence as set forth in SEQ ID NO: 116, 117, 118,119, 120, or 121 or a complementary sequence thereof together with asuitable probe for detecting presence or absence of a particularmutation. In some embodiments, a primer and probe of Table 3 is used. Insome embodiments, one or more PCR amplicons may be sequenced and theobtained sequence may be evaluated for purposes of detecting presence orabsence of a particular mutation. In some embodiments, a pair of primersdisclosed in Table 1, 2 or 5 may be used to amplify one or more regionsof the DYSF gene for purposes of determining the sequence of the DYSFand/or detecting presence or absence of a particular mutation in theDYSF gene.

In some aspects, the disclosure relates to the methods for detectingpresence of a pseudoexon peptide insertion in between exon 44 and exon45 of the DYSF gene (e.g., the peptide encoded by PE44.1). In someembodiments, the peptide insertion is detected by an immunoassay, suchas an ELISA or Western blot. However, other protein detection assays maybe used such as immunohistochemistry, immunocytochemistry,radioimmunoassays, or peptide sequencing by mass spectroscopy. In someembodiments, the immunoassay is probed with an anti-PE44.1 antibody.Antibodies against a mutant DYSF protein can target any region, forexample the N-terminus or C-terminus, of the protein. In someembodiments, an antibody (e.g., an anti-PE44.1 antibody) targets apeptide encoded by the PE44.1 pseudo exon. In some embodiments ananti-PE44.1 antibody targets the C-terminal portion of the peptideencoded by the PE44.1 pseudoexon. In some embodiments, an anti-PE44.1antibody targets SEQ ID NO: 116.

As used herein, the term “antibody” refers to an immunoglobulin, whethernatural or wholly or partially synthetically produced. All derivativesthereof which maintain specific binding ability are also included in theterm. The term also covers any protein having a binding domain which ishomologous or largely homologous to an immunoglobulin binding domain.These proteins may be derived from natural sources, or partly or whollysynthetically produced. An antibody may be monoclonal or polyclonal. Theantibody may be a member of any immunoglobulin class, including any ofthe human classes: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgGclass, however, are preferred in the present invention.

The term “antigen binding fragment” or “antibody fragment” refers to anyderivative of an antibody which is less than full-length. In someembodiments, the antibody fragment retains at least a significantportion of the full-length antibody's specific binding ability. Examplesof such fragments include, but are not limited to, Fab, Fab′, F(ab′)₂,scFv, Fv, dsFv diabody, and Fd fragments. The antibody fragment may beproduced by any appropriate means. For instance, the antibody fragmentmay be enzymatically or chemically produced by fragmentation of anintact antibody or it may be recombinantly produced from a gene encodingthe partial antibody sequence. Alternatively, the antibody fragment maybe wholly or partially synthetically produced. The antibody fragment mayoptionally be a single chain antibody fragment. Alternatively, thefragment may comprise multiple chains which are linked together, forinstance, by disulfide linkages. The fragment may also optionally be amultimolecular complex. A functional antibody fragment will typicallycomprise at least about 50 amino acids and more typically will compriseat least about 200 amino acids.

Single-chain Fvs (scFvs) are recombinant antibody fragments consistingof only the variable light chain (V_(L)) and variable heavy chain(V_(H)) covalently connected to one another by a polypeptide linker.Either V_(L) or V_(H) may be the NH₂-terminal domain. The polypeptidelinker may be of variable length and composition so long as the twovariable domains are bridged without serious steric interference.Typically, the linkers are comprised primarily of stretches of glycineand serine residues with some glutamic acid or lysine residuesinterspersed for solubility.

An Fv fragment is an antibody fragment which consists of one V_(H) andone V_(L) domain held together by noncovalent interactions. The termdsFv is used herein to refer to an Fv with an engineered intermoleculardisulfide bond to stabilize the V_(H)-V_(L) pair.

A F(ab′), fragment is an antibody fragment essentially equivalent tothat obtained from immunoglobulins (typically IgG) by digestion with anenzyme pepsin at pH 4.0-4.5. The fragment may be recombinantly produced.

A Fab fragment is an antibody fragment essentially equivalent to thatobtained by reduction of the disulfide bridge or bridges joining the twoheavy chain pieces in the F(ab'), fragment. The Fab' fragment may berecombinantly produced. A Fab fragment is an antibody fragmentessentially equivalent to that obtained by digestion of immunoglobulins(typically IgG) with the enzyme papain. The Fab fragment may berecombinantly produced. The heavy chain segment of the Fab fragment isthe Fd piece.

Cells

In some aspects, the present disclosure relates to the delivery ofantisense nucleic acids (e.g. oligonucleotides) to a target cell. Insome embodiments, the cell is of a subject having a DYSF gene containinga c.4886+1249 (G>T) mutation. The cell may be heterozygous for themutated gene or may be homozygous. The cell may have a second DYSF gene(a second allele of the gene) encoding a wild-type DYSF protein. Thecell may have a second DYSF gene (a second allele of the gene) having adifferent mutation than the c.4886+1249 (G>T) mutation. For example, thecell may contain a DYSF gene comprising a mutation that causes apremature stop codon (e.g., within a region encoding the C2D domain ofDYSF protein). For example, the premature stop codon may be ac.3444_3445delTGinsAA mutation. The cell to which the antisense nucleicacid is delivered may be in vitro or in vivo.

In some embodiments, the cell is a mammalian cell. In some embodimentsthe mammalian cell is a human cell. The cell may be from a subjecthaving a muscular dystrophy that is associated at least in part with theDYSF gene comprising the c.4886+1249 (G>T) mutation. For example, themuscular dystrophy is of the Miyoshi Myopathy-type, or other musculardystrophy caused by abnormal expression of the dysferlin gene product.In some embodiments, the cell is a myoblast or other muscle progenitorcell. In some embodiments, the cells are muscle cells. In someembodiments of the disclosure the muscle cells are striated (e.g.skeletal) muscle cells (e.g., myotubes). In some embodiments, the musclecells are cardiac smooth muscle cells. In some embodiments, the musclecells are smooth muscle cells. In some embodiments the cells are notmuscle cells. In some embodiments, cells are of the brain, heart,kidneys, lungs, uterus, spleen, pancreas or muscle tissue of a subject.In some embodiments, the cell is a non-human cell (e.g., a non-humanmammalian cell, e.g., a mouse cell). For example, the cell may be anon-human cell (e.g., a mouse cell) engineered to contain one or morecopies of the human DYSF gene comprising the c.4886+1249 (G>T) mutation.

Pharmaceutical Compositions

According to some aspects of the disclosure, compositions are providedthat comprise an agent (e.g., an antisense nucleic acid (e.g., anoligonucleotides) or vector comprising the same) and a carrier. As usedherein, the term, “carrier” refers to an organic or inorganicingredient, natural or synthetic, with which the active ingredient iscombined to facilitate an intended use.

For example, pharmaceutical compositions are provided that comprise anantisense nucleic acid and a pharmaceutically-acceptable carrier. Asused herein the term “pharmaceutically acceptable carrier” refers to acarrier that is suitable for pharmaceutical administration. The termpharmaceutically-acceptable carrier includes compatible solid or liquidfillers, diluents or encapsulating substances that are suitable foradministration to a human or other vertebrate animal.

Components of pharmaceutical compositions also are capable of beingcommingled with the agents of the present disclosure, and with eachother, in a manner such that there is no interaction that wouldsubstantially impair the desired pharmaceutical efficiency.Pharmaceutical compositions may include solvents, dispersion media,coatings, antibacterial and antifungal s, isotonic and absorptiondelaying agents, and other suitable components compatible withpharmaceutical administration. Supplementary active agents can also beincorporated into the compositions. Active ingredients (e.g.,oligonucleotides) may be admixed or compounded with any conventional,pharmaceutically acceptable carrier or excipient. Pharmaceuticalcompositions are generally sterile and prepared using aseptic technique.A sterile phosphate-buffered saline is one example of a pharmaceuticallyacceptable carrier. Other suitable carriers may be used. Pharmaceuticalcompositions also may comprise suitable solid or gel phase carriers orexcipients. Examples of such carriers or excipients include but are notlimited to calcium carbonate, calcium phosphate, various sugars,starches, cellulose derivatives, gelatin, and polymers such aspolyethylene glycols.

Antisense nucleic acids may be administered per se (neat) or in the formof a pharmaceutically acceptable salt. When used in medicine the saltsare generally pharmaceutically acceptable, but non-pharmaceuticallyacceptable salts may conveniently be used to prepare pharmaceuticallyacceptable salts thereof. Such salts include, but are not limited to,those prepared from the following acids: hydrochloric, hydrobromic,sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulphonic, tartaric, citric, methane sulphonic, formic, malonic,succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, suchsalts can be prepared as alkaline metal or alkaline earth salts, such assodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%vv/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the agents into associationwith a carrier that constitutes one or more accessory ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelybringing the agents into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct. Liquid dose units are vials or ampoules. Solid dose units aretablets, capsules and suppositories.

An effective amount, also referred to as a therapeutically effectiveamount, of an antisense nucleic acid (e.g. oligonucleotide) capable ofmodulating splicing in a cell in which the DYSF gene is expressed is anamount sufficient to ameliorate at least one adverse effect associatedwith expression, or reduced expression, of the gene in a cell or in anindividual in need of such modulation. The therapeutically effectiveamount to be included in pharmaceutical compositions may be selectedbased upon several factors, e.g., the type, size and condition of thepatient to be treated, the intended mode of administration, the capacityof the patient to incorporate the intended dosage form, etc.

In some cases, antisense nucleic acids may be prepared in a colloidaldispersion system. Colloidal dispersion systems include lipid-basedsystems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. An example of a colloidal system that may be used in methodsprovided herein is a liposome. Liposomes are artificial membrane vesselsthat are useful for delivering antisense nucleic acids in vivo or invitro. It has been shown that large unilamellar vesicles can encapsulatelarge macromolecules. Nucleic acids and other components (e.g., viralvectors) can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form. Liposomes may betargeted to a particular tissue by coupling the liposome to a specificligand such as a monoclonal antibody, sugar, glycolipid, or protein.Ligands which may be useful for targeting a liposome to, for example, asmooth muscle cell or skeletal muscle cell include, but are not limitedto: intact or fragments of molecules that interact with muscle cellspecific receptors and molecules, such as antibodies, which interactwith the cell surface markers. Lipid formulations for transfection arecommercially available from QIAGEN, for example, as EFFECTENE™ (anon-liposomal lipid with a special DNA condensing enhancer) andSUPERFECT™ (a dendrimeric technology). Liposomes are commerciallyavailable from Invitrogen, Life Technologies, for example, asLIPOFECTIN™, which is formed of cationic lipids such as N-[1-(2, 3dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) anddioleoyl phophotidylethanolamine (DOPE), as well as other lipid-basedreagents including Lipofectamine and Oligofectamine. Certain cationiclipids, including in particular N-[1-(2, 3dioleoyloxy)-propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), maybe advantageous when combined with the antisense nucleic acids (e.g.oligonucleotides) analogs of the disclosure.

In one embodiment, antisense nucleic acids may be formulated with abiocompatible microparticle or implant that is suitable for implantationor administration to a recipient. Bioerodible implants may include abiodegradable polymeric matrix, for example, for containing an exogenousexpression construct engineered to express an antisense nucleic acidunder the control of an appropriate promoter. The polymeric matrix canbe used to achieve sustained release of the therapeutic agent in thesubject. A polymeric matrix may be in the form of a microparticle suchas a microsphere, in which an antisense nucleic acid and/or othertherapeutic agent is dispersed throughout a solid polymeric matrix, or amicrocapsule, in which antisense nucleic acid and/or other therapeuticagent is stored in the core of a polymeric shell. Other forms of thepolymeric matrix for containing a therapeutic agent include films,coatings, gels, implants, and stents. The matrix composition also can beselected not to degrade, but rather, to release by diffusion over anextended period of time. In some embodiments, antisense nucleic acidsare administered to the subject via an implant while the othertherapeutic agent is administered.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver antisense nucleic acids (e.g, oligonucleotides) and/or theother therapeutic agent to a subject. Biodegradable matrices arepreferred. Such polymers may be natural or synthetic polymers. Thepolymer is selected based on the period of time over which release isdesired, generally in the order of a few hours to a year or longer.Typically, release over a period ranging from between a few hours andthree to twelve months may be used. A polymer may be in the form of ahydrogel, e.g., a hydrogel that can absorb up to about 90% of its weightin water and which is optionally cross-linked with multi-valent ions orother components, e.g., polymers.

Other exemplary compositions that can be used to facilitate uptake of anucleic acid include calcium phosphate and other chemical mediators ofintracellular transport, microinjection compositions, electroporationand homologous recombination compositions (e.g., for integrating anucleic acid into a preselected location within the target cellchromosome).

In addition to the formulations described previously, the agents mayalso be formulated as a depot preparation. Such long-acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

Other suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems.

Kits

Agents (e.g., antisense nucleic acids) described herein may, in someembodiments, be assembled into pharmaceutical or diagnostic or researchkits to facilitate their use in therapeutic, diagnostic or researchapplications. A kit may include one or more containers housing thecomponents and instructions for use. Specifically, such kits may includeone or more agents (e.g., antisense nucleic acids) described herein,along with instructions describing the intended therapeutic applicationand the proper administration of these agents. In certain embodimentsagents in a kit may be in a pharmaceutical formulation and dosagesuitable for a particular application and for a method of administrationof the agents.

The kit may be designed to facilitate use of the methods describedherein by physicians and can take many forms. Each of the agents of thekit, where applicable, may be provided in liquid form (e.g., insolution), or in solid form, (e.g., a dry powder). In certain cases,some of the compositions may be constitutable or otherwise processable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species (for example, water or a cell culture medium),which may or may not be provided with the kit. As used herein,“instructions” can define a component of instruction and/or promotion,and typically involve written instructions on or associated withpackaging of the disclosure. Instructions also can include any oral orelectronic instructions provided in any manner such that a user willclearly recognize that the instructions are to be associated with thekit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet,and/or web-based communications, etc. The written instructions may be ina form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, whichinstructions can also reflects approval by the agency of manufacture,use or sale for human administration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing agents described herein. The agents may bein the form of a liquid, gel or solid (powder). The agents may beprepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include the active agents premixed and shippedin a syringe, vial, tube, or other container. The kit may have one ormore or all of the components required to administer the agents to apatient, such as a syringe, topical application devices, or IV needletubing and bag.

The kit may have a variety of forms, such as a blister pouch, a shrinkwrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, ora similar pouch or tray form, with the accessories loosely packed withinthe pouch, one or more tubes, containers, a box or a bag. The kit may besterilized after the accessories are added, thereby allowing theindividual accessories in the container to be otherwise unwrapped. Thekits can be sterilized using any appropriate sterilization techniques,such as radiation sterilization, heat sterilization, or othersterilization methods known in the art. The kit may also include othercomponents, depending on the specific application, for example,containers, cell media, salts, buffers, reagents, syringes, needles, afabric, such as gauze, for applying or removing a disinfecting agent,disposable gloves, a support for the agents prior to administration etc.

EXAMPLES Example 1 Materials and Methods Patients.

Two Miyoshi myopathy patients (ID# 8597, 8601, designated herein as P1and P2, respectively) are siblings that exhibited typical MM diseasesymptom onset and progression. They do not express dysferlin in eithertheir muscles or monocytes and exome sequencing determined that they areheterozygous for a nonsense mutation in dysferlin exon 32(c.3444_3445delTGinsAA) that causes a premature stop codon within theC2D domain. Further sequencing analyses failed to identify a secondpathogenic allele, suggesting that this mutation lies within non-codingregions of the gene. Dermal fibroblasts and blood were collected fromthese patients and their immediate relatives for DNA analyses and thefibroblasts were used to develop myogenic cell culture lines.

DNAs from 112 unrelated patients representing 81 pedigrees clinicallydiagnosed with MM or LGMD2B, and from an additional 724 randomindividuals, (either clinically normal or with non-related conditions)were used in these studies. In the second phase of the study, samplesfrom eight unrelated MM patients who were DYSF negative in monocyteassays and for whom at least one of the pathogenic DYSF mutations hadnot been identified by exon sequencing, were tested.

Cell Cultures. Dermal Fibroblast Cultures.

Patient skin biopsies were obtained with informed consent and culturedas explants in RPMI Medium (RPMI1640 (GIBCO) or DMEM Medium (DMEMGlutamax with pyruv ate (GIBCO)), each containing 15% FBS (Sigma), 100units/ml penicillin, 100, μg/ml streptomycin and 0.25 μg/ml Fungizone(Gibco)) to generate fibroblast lines. These were later routinelypassaged in DMEM Fibroblast Growth Medium (GM) (DMEM, 15% FBS, 1×Non-Essential Amino Acids (GIBCO), 10 mM HEPES (GIBCO). Normal adulthuman dermal fibroblasts were obtained from Lonza, ATCC, and LifelineCell Technology (termed NHDF-1, −2, and −3, respectively).

Myogenic Conversion of Dermal Fibroblasts and Myoblast Lines.

Immortalized inducible fibroblast-derived myogenic (iFDM) cell cultureswere established from patient and normal NHDF fibroblast lines usingmethods described. Fibroblasts were transduced with atamoxifen-inducible MyoD lentivirus (Lv-CMV-MyoD-ER(T), Addgene). Thesecells proliferate as fibroblasts until induced to express MyoD by4-hydroxytamoxifen (TMX) treatment (5 μM TMX (Sigma, H-7904) in GM for 1day followed by 3 days in 1μM TMX in differentiation medium (DM) (DMEMGlutamax with pyruvate: Medium 199 (Gibco) (3:1), 2% horse serum(HyClone), 20 mM HEPES, and 20 μg/ml insulin, 11 μg/ml transferrin, 1.3μg/ml selenium (2× ITS, Gibco), replacing DM every third day. In initialexperiments TMX was added throughout the differentiation period, butequivalent differentiation was observed with 3 days of treatment, so theTMX induction in DM was shortened to 3 days. Some fibroblasts were alsoimmortalized by transduction with an hTERT lentiviral vector(Lv-CMV-hTERT-IRES-Puro, UCLA Vector Core) prior to myogenic conversionwith the MyoD lentivirus, extending the proliferative potential of thesecells. Applicants have also obtained a myoblast cell line (OlUbic-CT2(“UBic”)) derived from normal human biceps muscle and immortalized bytransduction with hTERT and CDK4 retroviruses to generate anunrestricted supply of ‘normal’ myogenic cells.

As shown in FIGS. 7 and 9, AON-mediated PE44.1-skipping can be inducedin the differentiated iFDM cells from patient fibroblasts (P1 and P2).In these experiments using standard and quantitative RT-PCR to evaluatemRNA transcripts, treatment with AONs (e.g., AONs 2 and 3) led to asignificant reduction of PE44.1 containing transcripts to ˜15-45% thatfound in control SCR or TE-treated cells, accompanied by a significant(˜50-100%) increase in the normal transcript levels. These resultsillustrate an in vitro model with which a functional impact ofAON-mediated DYSF PE44.1-skipping on patients' cells may be evaluated,for example.

Immunocytochemistry.

Differentiated iFDM cells were fixed with 4% paraformaldehyde then for10 min. then permeabilized and immunostained using anti -myosin heavychain antibody (MF20, monoclonal supernatant 1/10 dil., DevelopmentalStudies Hybridoma Bank) and detected with Alexa 488 anti-mouse IgG(Invitrogen) and Hoechst 33258 to stain nuclei with methods asdescribed.

Protein Analysis. Proteins were extracted from differentiated iFDM cellsusing RIPA buffer (40 mM Tris-HCl pH 8, 150 mM sodium chloride 1% TritonX—100. 0.5% sodium deoxycholate, 0.5% SDS) with protease inhibitors(cOmplete. Roche) then a sample was quantified using a BCA protein assay(Thermo) (Thermo) assay. The remaining protein was heated at 70° C. inSDS-PAGE Laemmli sample buffer 10 min, then 10 ug protein was separatedon 7.5% SDS-PAGE gels and blotted onto nitrocellulose filters using aniBlot Gel Transfer Device (Life Technologies, program P3 for 10 min.)and analyzed by western immunostaining using LI-COR Odyssey blockingreagent and methods. Primary antibodies included and anti-dysferlinspecific for the C-terminal end (NCL-Hamlet, Leica, 1/1000) orN-terminal end (Romeo, JAI-1-49-3, Abcam, 1/500), and anti-GAPHD(10R-G109a Fitzgerald, 1/1000). Blots were quantitatively analyzed usinga LI-COR Odyssey infrared imager.

Nucleic Acid Purification And Reverse Transcription.

Genomic DNA and RNA were prepared from cells using Gentra Puregene(Qiagen) and TRIzol (Life Technologies) reagents, respectively andmanufacturers protocols. RNA was DNAse digested (TURBO DNA-Free,Ambion), to remove DNA contaminants then 0.2-2 ug RNA was reversetranscribed (High Capacity cDNA Reverse Transcription Kit (AppliedBiosystems)) using manufacturers protocols.

PCR Amplification.

For RNA and cDNA analyses used DYSF mRNA variant 8 (NM_003494.3) wasused as the reference sequence, as it is the predominant isoform inskeletal muscle. PCR primers were used to amplify and sequence the cDNAfrom patient and normal iFDM cells to determine whether the exon 32(c.3444_3445delTGinsAA) mutant allele is expressed in these cells. Thefollowing primers were used: DYSF31-F (5′-GTGTGAACAGACCCACGAT-3′) andDYSF33-R (5′-GTCGTACAGCTCCACCACAA3′).

In some embodiments, to facilitate sequencing of a complete DYSF cDNA, aPCRTiler v1.42 program was used to design 17 primer sets to amplify ˜500bp cDNA segments that overlapped by ˜50 bp and spanned the entire 6.9kbDYSF cDNA (Table 1). cDNAs from cell cultures (corresponding to 40 nginput RNA) was amplified by PCR using Hotmaster Taq DNA polymerase (5PRIME) as follows: 95° C. for 5 min., 30 cycles of 95° C. for 30 sec.,59.1° C. for 30sec., 72° C. for 1 min., then 72° C. for 10 min. PCRproducts were analyzed by gel electrophoresis and amplified productswere isolated and sequenced.

Similarly primers were designed (Table 2) to amplify through andsequence DYSF intron 44i using 20 ng of genomic DNA from patient cellsand blood samples. The same PCR conditions were used.

Allelic Discrimination Assays.

TaqMan Probes were designed (Applied Biosystems) to distinguish normaland mutant alleles of the novel point mutation in intron 44i of the twoMM patients (Table 3). These were used in PCR reactions (TaqmanGenotyping Master Mix Kit, Applied Biosystems) to amplify 10 ng ofgenomic DNA from the two MM patients, their immediate family members,112 unrelated patients representing 81 pedigrees with individualsclinically diagnosed with MM or LGMD2B. Of these, 16 pedigrees had DYSFmutations defined in both alleles and 3 had a mutation defined in onlyone allele. 724 DNA samples from a random population of normalindividuals and those with unrelated diseases were also screened. PCRconditions were: 95° C. for 10 min., then 50 cycles of 92° C. for 15sec., 60° C. for 1 min. PCR products were analyzed on a Bio-Rad CFX384Touch Real-Time PCR Detection System using the allelic discriminationsoftware.

Antisense Oligonucleotide Transfections.

The 44i mutation identified leads to inclusion of an in-frame pseudoexonthat disrupts the normal DYSF protein sequence. Inhibiting the splicingof PE44.1 allows restored synthesis of normally spliced DYSFtranscripts. AONS targeting ESE sequences within PE44.1 were designedthat could enhance its inclusion in spliced mRNA, along with anon-specific scrambled AON that does not target this region (Table 4).These AONs were synthesized as 2′-O-methyl RNA with full-lengthphosphorothioate backbones (Integrated DNA Technologies). iFDM cellsfrom patient P1, P2 and normal NHDF-2 fibroblasts were allowed todifferentiate for 6-9 days to form myotubes. Cells were transfected witheach AON (600 nM) (or TE buffer as control) using Oligofectamine (LifeTechnologies) and the manufacturer's protocol. At specific times afterAON addition (1 to 8 days), RNA was extracted and expression of wildtype and PE44.1 containing mutant RNA evaluated by RT-PCR using primersthat distinguish the two mRNA forms based on amplicons size. For thisprimers were: DYSF44/45-F -and DYSF44/45-R (Table 5) 5′, which generatea 143 bp amplicon from the normal cDNA (exon 44+45) and 320 bp ampliconsfrom the mutant cDNA containing PE44.1 (exon 44+PE44.1+45). cDNAs(corresponding to 20 ng input RNA) were amplified using Hotmaster TaqDNA polymerase as follows: 94° C. for 2 min., 30 cycles of 94° C. for 20sec., 58° C. for 20 sec., 65° C. for 1 min., then 65° C. for 10 min. PCRproducts were analyzed by gel electrophoresis.

For quantitative PCR (Q-PCR), cDNAs (corresponding to 20 ng input RNA)were amplified using primers (Table 5) that distinguishPE44.1-containing mutant (DYSF 44.1-Q F and R) RNA and normal DYSF44/45-Q F.2 and R.2). Primers for GAPDH were used to normalize RNAlevels. Primers specific to sequences spanning the junction of exons 50and 51, present in all DYSF forms, were used to determine the total DYSFmRNA levels, and this served as the 100% DYSF expression value toapproximate the relative expression of the mutant and normal exon 44/45splice forms. For Q-PCR, DyNAmo HS SYBR Green qPCR Kit reagents wereused (Thermo Scientific) and a Bio-Rad CFX384 C-1000 Touch Real-Time PCRDetection System. The PCR conditions were 95° C. for 15 min., 50 cyclesof 94° C. for 10 sec., 58° C. for 30 sec., 72° C. for 30 sec., then 72°C. for 10 min. followed by melt curve analysis (65-95° C.) to ensureproduct quality.

Statistics. Statistical significance was evaluated using one-way ANOVAwith post-hoc Tukey tests. Prism 5.0 statistical analysis software(GraphPad Software) was used.

Example 2 A Novel Deep Intronic Mutation in Dysferlin Leads toExpression of a Pseudoexon Miyoshi Myopathy Patients: Restoration ofNormal DYSF mRNA Mediated by Antisense Oligonucleotides Identificationof a Deep Intronic Mutation in DYSF Intron 44.

The two MM patients in our study, P1 and P2, do not express dysferlin ineither their muscles or monocytes and exome sequencing determined thatthey are heterozygous for a nonsense mutation in dysferlin exon 32(c.3444_3445delTGinsAA) that causes a premature stop codon within theC2D domain. Further exome sequencing analyses failed to identify asecond pathogenic allele, suggesting that this mutation lies withinnon-coding regions of the gene. To identify the other mutant allele, amyogenic cell culture system was established with cells from thepatients and normal controls. Myogenic cell lines were generated fromdermal fibroblasts cultured from patient skin biopsies. The fibroblastsfrom patients, along normal control fibroblasts, were converted toinducible fibroblast-derived myogenic (iFDM) cell cultures by usinglentiviral constructs to introduce a tamoxifen-inducible form of themyogenic regulator MyoD, which drives the myogenic program. In additionlines were immortalized by introducing lentiviral hTERT to extend theirproliferative lifespan. Upon treatment of cells with TMX, resultingcultures contained numerous multinucleate myotubes and expresseddifferentiated muscle proteins such as myosin heavy chain as shown inFIG. 1A, and on very rare occasion were observed to spontaneouslycontract. These lines thus provide an unrestricted supply ofpatient-derived myogenic cells with a high capacity to differentiate.

Western blots containing protein from patient-derived iFDMs probed withC-terminal anti-dysferlin antibodies (NCL-Hamlet) showed low levels ofdysferlin protein of apparently normal size (at approximately 20-40% theamount in normal iFDMs) (FIG. 1B). Similar results were obtained withanti-N-terminal dysferlin antibody (Romeo). Therefore, though dysferlinprotein was not detected in muscles or monocytes from these patients,low levels of dysferlin protein are produced in these mutant cellcultures. The known mutation in these patients that causes a prematurestop codon within exon 32 and could generate a truncated protein. Amutation in the second DYSF allele allows the synthesis of some proteinof normal or near-normal size.

cDNA was prepared using RNA from differentiated iFDM cells from derivedpatients P1, P2 and normal control NHDF-2 fibroblasts. PCR primers wereused that amplify the cDNA region around the known exon 32 mutation todetermine whether transcripts carrying this mutation are expressed inthe patient-derived iFDMs. Sequence analysis of amplified RT-PCRproducts showed that RNA did not contain exon 32 nonsense mutantsequence but rather contained only the wild-type sequence in thisregion, indicating that the DYSF mRNAs are produced only from the otherunknown allele. The products of the exon 32 mutant allele are thereforelikely degraded due to rapid nonsense-mediated decay mechanisms. Todefine the structure of the mRNA produced in the mutant iFDMs, PCRprimer sets that generated 17 overlapping amplicons (Table 1) were usedto sequence the 6.9 kb DYSF cDNA from these cells. Most of these primersets generated amplicons of the same size using iFDM cDNAs from patientsP1, P2 and normal cells (FIG. 2A, panels A, D). However, two of theoverlapping primer sets revealed a novel amplification product only inpatient cDNAs (FIG. 2A, panels B, C). Using additional PCR primers thatflank this 44/45 junction, cDNAs from skeletal muscle biopsy tissue RNAwere amplified from Patient 1 and an unrelated, non-symptomaticindividual. As shown in FIG. 2B, very low amounts of DYSF transcriptsare present in the patient's muscle tissue and include both mutant andnormal splice forms as detected by the two amplification products (320and 143 bp, respectively). Only the normal cDNA amplification product isobserved in the normal control muscle.

Subsequent sequence analysis of these amplicons from each patient andcontrol cDNAs revealed that the novel amplicons contained 177bp ofintron 44i sequence spliced into the cDNA at the exon 44 exon 45junction, maintaining the normal reading frame (FIG. 3). As a result, 59amino acids are inserted into the protein sequence. The patientstherefore express a novel pseudoexon (PE), termed PE44.1, derived fromsequences within DYSF 44i that are spliced into the mature transcript.

DYSF 44i region was sequence using genomic DNA from iFDMs and bloodsample from both patients and compared it with the genomic NCBIreference sequence. Sequence analysis revealed a point mutation in bothpatients nearly midway in intron 44i (c.4886+1249 (G>T)) (FIG. 4A, B).This (G>T) mutation, which occurs 2 bp after the 3′ end of the PE44.1sequence, generates a consensus splice donor sequence at that site (FIG.4B) that promote the aberrant splicing of PE44.1 into the maturetranscript. Along with the novel splice donor site created by this (G>T)mutation, analysis of the intronic sequence upstream of PE44.1 revealsthat other sequence elements required for splicing are also present inthis region. As shown in FIG. 4C, there is a conserved splice acceptorsite at the 5′ end of PE44.1 along with a required pyrimidine-richregion immediately upstream and two potential lariat branch pointsequences (as identified by Human Splicing Finder version 2.4.1,typically 2-50 bp upstream of the splice site). Therefore a multipleelements required by the splicing machinery are in place within thisintronic region to allow the aberrant splicing of PE44.1 within the mRNAin the presence of this (G>T) point mutation.

In addition to the (c.4886+1249 (G>T)) mutation, 4 common sequencevariants were identified in DYSF cDNA in patients P1 and P2 relative tothe reference sequence (Table 6). Sequence analysis of DYSF intron 44iidentified 6 additional polymorphisms (Table 7). All of these arecommon, previously identified polymorphisms not linked to disease.

The alternate DYSF splice forms present in patients P1 and P2 cells areshown in FIG. 5A. Our cDNA amplification and sequencing results showthat both normal and mutant PE44.1 mRNA splice forms are present inpatient iFDM cells, typically present in approximately equal proportions(FIGS. 2, 7, 9). The impact of PE44.1 inclusion in the DYSF mRNA andprotein is further shown in FIG. 5B and C. DYSF exons 44-45 encode partof the conserved C2F domain of dysferlin. Insertion of the PE44.1sequence leads to the in-frame insertion of 59 amino acids within theC2F domain and a large disruption of this domain that wouldsignificantly impair protein function. This 59 amino acid sequence isunique and not homologous to any other peptide or protein structure.

Pedigree Analysis.

Genomic DNA was amplified and sequenced from blood samples taken fromimmediate relatives of P1 and P2 to determine the inheritance of allsequence variants in DYSF 44i, as well as the DYSF exon 32 mutationpreviously identified in these patients. FIG. 6 shows the DYSF genotypesof the family, revealing the maternal inheritance of the DYSF 44imutation (c.4886+1249 (G>T)) responsible for PE44.1 expression. Thesegregation of all DYSF intron 44i allelic variants in family members isshown in Table 7.

Prevalence of the Deep Intronic DYSF (c.4886+1249 (G>T)) Mutation.

An allelic discrimination assay was used to screen genomic DNA from 112patients (81 pedigrees) clinically diagnosed with either MM or LGMD2B,along with 724 individuals from a random population (either normal orwith an unrelated disease), using the patient P1, P2 and their 4 familymembers as a reference. For this, TaqMan primers and probes weredesigned that distinguish the (c.4886+1249 (G>T)) point mutation fromthe normal genomic sequence in DYSF 44i. The 2 patients and 2 familymembers carry this mutation.

In the second phase of the study, separate analysis of eight suspecteddysferlinopathy patients that had only one or neither of theirpathogenic DYSF mutations identified by exon sequencing. These patientswere screened for the C.4886+1249 (G>T) mutation. As shown in Table 9,DYSF protein was low or absent in these patients, with serum CK, anindicator of muscle damage, elevated in most. Of the eight patientsscreened, two carried the c.4886+1249 (G>T) variant allele.

Antisense Oligonucleotide-Mediated Pseudoexon PE44.1 skipping.

The inclusion of PE44.1 leads to a disruptive insertion within thedysferlin protein; therefore preventing the spicing of this pseudoexoninto the mature mRNA will promote the synthesis of normal mRNA. AONsdirected against three possible exonic splice enhancer sequences withinPE44.1 were designed (Table 4, FIG. 7A) to block these enhancersequences and reduce the amount of PE44.1 included in maturetranscripts. These AONS were transfected into patient and normal controliFDM cells and mRNAs were analyzed two to eight days later. As shown inFIG. 7B, AON1 treatment for 2 days did not detectably affect the levelsof the mutant or normal transcripts and was similar to controls treatedwith TE or a non-targeting scrambled AON. However, AON2 and AON3,reduced the relative levels of the mutant splice form containing PE44.1,and led to an increase in the levels of the normal mRNA splice form incells from both patients P1 and P2 (FIGS. 7B-D). For example, as shownin FIGS. 7C and D, the PE44.1 mutant form of RNA representedapproximately 32-34% of the DYSF mRNA in P1 control cultures (TE or SCRscrambled control oligo), and approximately 41% in P2. AON2 treatmentfor 2 days reduced this mutant form to ˜12% and 7% of the total DYSFlevels in P1 and P2, respectively, while AON3 reduced it to ˜15% and 6%(P1, P2 respectively). Concomitantly, there was an increase in thenormal transcript levels in these cells: AON2 increased the normal formfrom ˜45% of DYSF mRNA to ˜70% in P1 cell, and from 38-44% to 78% in P2cells. AON3 increased the normal form from ˜45% of DYSF mRNA to ˜66% inP1 cell, and from 38-44% to 92% in P2 cells. As expected, no PE44.1containing mRNA was expressed in normal control cells. These resultsdemonstrate that AONs can significantly modify mRNA splicing to inhibitmutant PE44.1 inclusion, which could potentially restore normal DYSFprotein levels and function in these cells.

Treatment of patient iFDMs with AON3 induced the synthesis of DYSFprotein as well as higher levels of normal DYSF transcripts that don'tcontain PE44.1 (FIGS. 8 and 9). Here, TMX-induced iFDM cells weretreated with AONs after 6 days of differentiation, at a point when cellsare undergoing fusion to form myotubes in these cultures. Eight daysafter adding AON3, there was a significant increase in DYSF proteindetectable by Western blotting (FIGS. 8A-8B). After one day of AON3treatment, there was a dramatic effect at the RNA level, with reducedexpression of the mutant PE44.1 form and increase in the normal spliceform (FIG. 9A, B). However, there was no significant change in theabundance of DYSF protein at this time (FIG. 8A, B). After three days ofAON3 treatment, DYSF protein levels were significantly higher in cellsfrom both patients, while normal control cells showed no differences dueto AON3. These elevated DYSF protein levels persisted through 8 dayspost-AON3 addition (5 days after the AONs were removed on day 3). Theeffects of AON3 on the relative abundance of the PE44.1 and normalsplice forms were also observed through 8 days post-AON3 addition (FIG.9A, B). Therefore, AON-mediated skipping of PE44.1 can restore morenormal levels of both DYSF mRNA and protein in these mutant cells.

Example 3 Production of an Anti-PE44.1 Antibody

Antibodies directed against the mutant peptide encoded by PE44.1havebeen produced. An immunizing peptide having the amino acid sequenceCAFEGEPALASDELPPSRDWR (SEQ ID NO: 122), corresponding to the C-terminalend of the 59 amino acid peptide encoded by PE44.1 (FIG. 3) wassynthesized. Antibodies were produced in two rabbits, both of which hadstrong titers against the immunizing peptide. Western blot analysis ofcultured myogenic iFDM cells from dysferlinopathy patients P1 and P2(that carry the c.4886_1249 (G>T) dysferlin mutation) shows that theanti-PE44.1 antibodies detect a band near the size of dysferlin inpatient but not in normal control cells (FIG. 10). Thus, the mutant formof dysferlin containing the extra PE44.1 encoded peptide is expressed inpatient cells and can be identified using specific antibodies.

Example 4 Method of Detecting DYSF Mutation in Patient Samples

RNA from whole blood of patients (with pathologies unrelated todysferlinopathy) were purified using the Qiagen PAXgene Blood RNA tubesand PAXgene Blood RNA Kit. Mutations in introns 44i were identifiedusing the primers described above. RT-PCR was used to amplify DYSF cDNAfrom the blood RNA samples. Amplicons for each of the DYSF primer pairswere produced from the blood RNA, with products that includedalternative DYSF mRNA isoforms known to be expressed normally in blood.These alternate isoforms arise from variant splicing of DYSF exons ordifferent promoter usage. These results demonstrate screening of patientblood cells to identify intronic DYSF mutations that are not readilyidentifiable using standard methods of exon sequencing of genomic DNA.Additionally or alternatively, biological samples (e.g., blood) obtainedfrom patients can be screened by Western blot using an anti-PE44.1antibody, as shown in Example 3 (FIG. 10).

TABLE 1 Primers (forward (F) and reverse (R)) used togenerate overlapping amplicons that span the entire dysferlin cDNA.Primer Name Sequence SEQ ID NO:  1F AGGTGCAAAATGCCGTGT 1  1RTTCACCCCTGCAAACACC 2  2F CACACCCGACACCGACAT 3  2R CTCCGCCTCATCTCCAGTG 4 3F CGACTCTGCCTGACCTGGA 5  3R AATGGTGCCCACGTCCAT 6  4FTCGTTCTCTCAGGACAGATGC 7  4R CTGAGGGTTGGCCGTCTT 8  5F GACCCCTTTGTGGAGGTCA9  5R GCTCCACCAGCTTGGTCTC 10  6F GGGGGAAGGTGTGGCTTAT 11  6RCAGCGAGTCCACGTCCTC 12  7F CCAGCTGCTTGGGATTGC 13  7R TCCCACAATTCTTGCCACA14  8F GCCCACCAAGTCCTCTTCTC 15  8R AAGCCGGGTCTGGTTCTC 16  9FTCACCTGAGCTTCGTGGAA 17  9R TTCTCCAGTGGCTCCATG C 18 10FCCACCTCGAGTACCGCAAG 19 10R CGTACAGCTCCACCACAATG 20 11FAACACCCTTAACCCCACCTG 21 11R CGGAGGTTCCTGATGACACA 22 12FCCCCAGCCTCGTGGTAGA 23 12R ACCTTCAGGGTGTCAAAATCC 24 13FTGCCTCCATAGGGGAGAGG 25 13R TGCAGGTCAGCTCGAACA 26 14F TGGAGCCCGTATTTGGAA27 14R TGCAGGGGGCTGTAGAGG 28 15F CGTCTGGCTCTGCATGTG 29 15RCCACTCGTGCTGGGATTTT 30 16F CTGCCAGCTGAGCAAGTCTG 31 16RGCCGCCACAGGATGAACT 32 17F CCGACACCTCCTTCCTGTG 33 17RTTGTGGTTCCAACTGTTTTATACTGA 34

TABLE 2 Primers (forward (F) and reverse (R)) used togenerate overlapping amplicons that span dysferlin intron 44. SEQ IDPrimer Name Sequence NO: DYSFi44.1F CCTGGGTGACAGAGCAAAAC 35 DYSFi44.1RGCCTAAACAAGCTCACATCCA 36 DYSFi44.2F TGAAATCTGAGAACAAGGAAAGGA 37DYSFi44.2R AATGGAAGGGTTTCTGTTGTGA 38 DYSFi44.3F CCTCCCATGCCTGTTTCC 39DYSFi44.3R TATACACACACAACTGCATCCAAAGA 40 DYSFi44.4FACCCAGTCTCAGGCCATAACC 41 DYSFi44.4R AGGGCCCTTCCTTCCCTA 42 DYSFi44.5FATGTCTGTGTCCATG TGT CTGC 43 DYSFi44.5R ATCTGAGAACTCCATCTACCATGT G 44DYSFi44.6F TCACCTGCAGGGAGCTCA T 45 DYSFi44.6R GGAAGGGGGACAGGACTTT 46DYSFi44F TTGGAGTCCCCCAATTTACC 47 DYSFi44R GCAAATGCATCTTCTGAGCTT 48

TABLE 3 TaqMan probes and primers used in allelic discrim-ination assays to screen genomic DNA forthe dysferlin intron 44 mutation (c.4886 + 1249 (G > T)). Primer NameSequence (5′ > 3′) SEQ ID NO: DYSFi44.1F GATGAGCTCCCACCAAGCA 49DYSFi44.1R AGCCCCTGGAACTCATACAGA 50 Probe Reporter Probe 1CACCTACCACTTCCCTCCA 51 (normal) (VIC labeled) Reporter Probe 2ACCTACCACTTACCTCCA (mutant) (FAM labeled) 52

TABLE 4AONs targeting human exonic splicing enhancer sequences (ESE) in DYSF PE44.1.AONs are 2′-O-methyl RNA with full-length phosphorothioate backbones.Target Sequence (sense strand) AON AON Sequence (5′ - 3′) (5′ - 3′)DYSF44.1 AON1 GAACAGUAGGUGUCUUCUGCAG CTGCAGAAGACACCTACTGTTC(SEQ ID NO: 53) (SEQ ID NO: 54) DYSF44.1 AON2 CCAGGGCUGGUUCACCUUCAAATTTGAAGGTGAACCAGCCCTGG (SEQ ID NO: 55) (SEQ ID NO: 56) DYSF44.1 AON3CUCCAAUCCCUGCUUGGUGG CCACCAAGCAGGGATTGGAG (SEQ ID NO: 57)(SEQ ID NO: 58) SCR (scrambled neg. ACGGCGUGACUAGUGGUGAG control)(SEQ ID NO: 59)

TABLE 5 Primers (forward (F) and reverse (R)) used in RT-PCR assays to analyze RNA for expression for thedysferlin intron 44 mutant and normal transcripts. PCR Primer NameSequence (5′ > 3′) DYSF 44/45-F TAACTACATCCCCTGCACGC (SEQ ID NO: 60)DYSF 44/45-R CGACCGTCTCACCGATCTTT (SEQ ID NO: 61)Quantitative PCR Primer Name Sequence (5′ >3′) DYSF 44.1-Q-FAGTGAGGACCCAGTCTCCTT (SEQ ID NO: 62) DYSF 44.1-Q-R AGCTCGAACATCCTCCAATCC(SEQ ID NO: 63) DYSF 44/45-Q-F.2 GCCCGTATTTGGAAAGATGT (SEQ ID NO: 64)DYSF 44/45-Q-R.2 CCGTCTCACCGATCTTTTCG (SEQ ID NO: 65) DYSF 50/51-Q-FCGTGCATTATCGTTCCCTGG (SEQ ID NO: 66) DYSF 50/51-Q-RAGGCATCCTTCTTGGCAATGG (SEQ ID NO: 67) GAPDH-Q-F ACCACAGTCCATGCCATCAC(SEQ ID NO: 68) GAPDH-Q-R ACCACAGTCCATGCCATCAC (SEQ ID NO: 69)

TABLE 6 cDNA sequence variants in Miyoshi myopathy patients P1 and P2.SEQ SNP Genotype Genotype NT AA  Global Chromosomal ID name Ref. Var.8597(P1) 8601 (P2) Type position position Exon Sequence Primers MAFlocation NO rs23 T C C|C C|C synony- c.1827 p.(D609=) 20 ATGT  6F + RT = 2: 7178 70 0359 mous T > C GGAT 0.4132/ 0215 6 GA[T/ 900 C]GC CATCCAGT rs22 A T T|T T|T synony- c.2583 p.(S861=) 25 TTGG  8F + R A =2: 7179 71 8835 mous A > T GCTC 0.4463/ 5152 5 TC[A/ 972 T]GT GGAT GAGArs23 C A A|A A|A synony- c.4008 p.(I1336=) 38 GTGC 11F + R A = 2: 718372 0360 mous C > A TTAG 0.4784/ 8597 6 AT[C/ 1042 A]CT GGCA TGGG rs62 GA A|A A|A synony- c.4731 p.(E1577=) 43 GACC 13F + R A = 2: 7188 73 1459mous G > A CCAG 0.0101/ 6100 39 GA[G/ 21 A]TG CTTG GTCC — TG AA TG|AATG|AA Indel c.3444_ p.(Y1148X) 32 TTGG 10F + R 2:7181 74 3445 TGAA7342-3 delTGi GA[T/ nsAA A][G/ A]GG AACC GCTA

With reference to Table 6, reference (Ref.) and variant (Var.) allelesfound in patients P1 and P2 and reported in the dbSNP database areshown. None of these SNP variants cause amino acid changes. The indel inexon 32 (c.3444_3445delTGinsAA) is a pathogenic mutation. The globalminor allele frequency (MAF) and chromosomal location (GRCh37/hg19assembly) are indicated, along with primer sets used to amplify andsequence these regions.

TABLE 7Genomic DNA sequence variants in DYSF intron 44 of Miyoshi myopathy patientsP1 and P2 and relatives. SEQ 8597 8601 Global Chromosomal ID SNP nameRef. Var. (P1) (P2) 8599 8600 8602 8603 nt position Sequence Primers MAFlocation NO novel G T G|T G|T G|T G|T G|T G|G c.4886 + 12 GGA i44.2F + RT = 0/0* 2: 718890 75 49G > T TTG 30 GAG G[G/ T]A AGT GGT AGG T rs379182A G G|G G|G G|G G|G G|G G|G c.4886 + 13 CTG i44.2F + R A = 0.1382: 718891 76 5 20A > G GCG 7/301 01 CAG G[A/ G]C CTC AGG CTA rs379182 CG G|G G|G G|G G|G G|G G|G c.4886 + 13 AGT i44.2F + R C = 0.172 2: 71889177 6 75C > G GGG 2/374 56 TGG G[C/ G]G TGT GTC AGG rs230359 C T T|T T|TC|T C|T T|T T|T c.4887 − TAC i44F + 13R C = 0.374 2: 718912 78 8162C > T ACA 7/816 36 CAC A[C/ T]TC AGG CCC AG rs230359 C T T|T T|T T|TT|T T|T C|T c.4887 − TTG i44F + 13R C = 0.254 2: 718913 79 9 73C > T GGT4/554 61 GCC C[C/ T]GT GTT GGC TG rs556981 A G G|G G|G A|G A|G G|G G|Gc.4887 − TAG i44.5F + R A = 0.292 2: 718901 80 53 11991A > G GGA 9/63799 AGG A[A/ G]G GGC CCT GCC rs379182 C G G|G G|G C|G C|G G|G G|Gc.4887 − CTC i44.5F + R C = 0.400 2: 718903 81 7 1083C > G ATG 4/871 15TGC C[C/ G]A CCA CCG CTG

With reference to Table 7, genomic variations within DYSF intron 44 inpatients P1, P2 and relatives are listed. The novel mutation c.4886+1249(G>T) results in PE44.1 inclusion in mature mRNAs. *The global MAF forthe novel variant is calculated based on our screen of 836 unrelatedindividuals, in which 0 additional individuals were found with thisvariant. Other variations are reported in the dbSNP database and havenot been shown to be pathogenic. The primer sets used to amplify andsequence these regions are shown.

TABLE 8Additional AONs for enhancing expression of dysferlin mRNA and protein.SEQ SEQ ID Target Sequence (sense strand) ID AON AON Sequence (5′ - 3′)NO: (5′ - 3′) NO: DYSF44. CACCUACCACUUACCUCCAA 82 GATTGGAGGTAAGTGGTAGG99 1 AON4 UC TG DYSF44. CCUCUGCAUCCUCCCCUCUC 83 CAGAGAGGGGAGGATGCAG 1001 AON5 UG AGG DYSF44. GCGAGAGAAAUAGCUGCCAC 84 GTGGCAGCTATTTCTCTCGC 1011 AON6 DYSF44. AUACAGAGUUCCUCUCUCCC 85 GGGAGAGAGGAACTCTGTAT 102 1 AON7DYSF44. CAGCAAUUCAGGAUGCCUAG 86 TTCCTAGGCATCCTGAATTG 103 1 AON8 GAA CTGDYSF44. GCCUAAACAAGCUCACAUCC 87 TTTGGATGTGAGCTTGTTTA 104 1 AON9 AAA GGCDYSF44. AUAGCUGCCACCCUCCCUCU 88 AGAGGGAGGGTGGCAGCTA 105 1 AON10 TDYSF44. GUCUUCUGCAGGUGCGAGA 89 TTTCTCTCGCACCTGCAGAA 106 1 AONII GAAA GACDYSF44. UAUGUGACUUGAUGACAUC 90 TCCGATGTCATCAAGTCACA 107 1 AON12 GGA TADYSF44. CAGACCCCGGUUUAUAAGUG 91 TTGCACTTATAAACCGGGGT 108 1 AON13 CAA CTGDYSF44. GUGAGUGACCACAACUAACU 92 TTGAGTTAGTTGTGGTCACT 109 1 AON14 CAA CACDYSF44. GGAGACUGGGUCCUCACUGC 93 CCTGCAGTGAGGACCCAGTC 110 1 AON15 AGG TCCDYSF44. UUCAAAAGCUGCAUUUCCCC 94 TTGGGGAAATGCAGCTTTTG 111 1 AON16 AA AADYSF44. UGGGAGCUCAUCACUGGCCA 95 CCCTGGCCAGTGATGAGCTC 112 1 AON17 GGG CCADYSF44. AGCUCGCCAGCCCCUGGAAC 96 TGAGTTCCAGGGGCTGGCGA 113 1 AON18 UCA GCTDYSF44. GAGGUCCUGCGCCAGCCCCU 97 TCCAGGGGCTGGCGCAGGAC 114 1 AON19 GGA CTCDYSF44. CUGCCCCAUCAGCAUUAGCC 98 TCAGGCTAATGCTGATGGGG 115 1 AON20 UGA CAG

TABLE 9 Dysferlin allele variants and the relative dysferlin expressionin eight patients Dysferlin expression in Patient Age of monocyte No.onset CK levels Allele 1 Allele 2 assay¹ JF01 28  3369 c.1834C > Tc.4886 + 1249 Absent (p.Q612X) G > T JF14  3  300 — — Absent JF15 18 198 c.3534C > T — Absent (p.I1178) JF19 18 10000 c.2997G > T — Absent(p.W999C) JF32 17  2800 — —  9% JF35 20  1800 c.857T > A c.4886 + 1249Absent (p.V286E) G > T JF67 32 28000 c.5341G > A — Absent (p.G1781R)JF85 17  5675 c.2997G > T — 10% (p.W999C) ¹The relative expression ofdysferlin in monocytes is presented as the proportion of the level innormal control samples.

ADDITIONAL SEQUENCES >DYSF Pseudoexon 44.1: SEQ ID NO: 116AAGACACCTACTGTTCCGATGTCATCAAGTCACATATTGCACTTATAAACCGGGGTCTGTTGAGTTAGTTGTGGTCACTCACCCTGCAGTGAGGACCCAGTCTCCTTGGGGAAATGCAGCTTTTGAAGGTGAACCAGCCCTGGCCAGTGATGAGCTCCCACCAAGCAGGG ATTGGAG> DYSF Intron 43i through intron 45i in the context of Homo sapienschromosome 2, GRCh38 Primary Assembly, NCBI Reference Sequence:NC_000002.12: SEQ ID NO: 117GTAACTTTCCTAGAGCCCTCACCTCCCCCAGAGTAGCAGGCTCAGGTACAAGTGGCCTATAGAACCTGGACACAAACTCTGCCTCAGGGAGTTCATAGTAGGTTGGGAAACAGACAAACACACAAAACTGAGAGGTGCCTGGATGGAGTTGTGTTAAGGACCAAGTGCTCTAGAAGGTCAGGGAAGGCCAGTGTCAGTACAGGACTTTGGGAAAATGGGGAAGGCTTCCTGGAAGGAATAGGACTCTTAGGATAGATGAGATTTTGATAGGCTGGGAGGGAGAAGAAAATAGTGTTTTAAGAGGGGCCAAAAGCATAGGCAAAGGTTTGAAAGAAAATTGCTTAATGTGTGTCTAGAACAAGAATAAAGCAGAAAGATAAGTAAGGCTGAGGGTATAGGTTGGGACTCTCAATGGTTAATGGGGAGGAGTGATGGGGGAAAGGAGCAGCGTTAGTGACTGCTATGCCTAGTCATGGCAGAGTCCCAGGTAAAAGGGAATGCTGGTAACACTCACCCGGTCTTTATTTAAAATTTTGATATTTTGTTCATCATGGACTATTGACACTGATTTTAATTTTTAAAATATTTCATTAAAGTACTGTTTATCTTAATAACTATTTTGGCCCTTCCTTACATTTTGTGCCCAAGATAAGTGCGTGAGCTGTCTCCCTCTAGTCATTGCCTGAGATGGAGAGGGGAGATTTGAAGGAGGAAAGGAGTCTGGAGTTATGGGGAGGTGACAGAGGTGGGAGGCAGAGTAATGTTGGGAAGAGAAAGAAAGAGGAAAGAAGAGAGGCAGGGGTGGGGCCTGATTTGCCTTTACAGGCATGAGGCTAGGGTCCCCTCTTGTCTCCTGAGGCAGGTTTAGGCCCAGCATGTGTCCTTCAGGTGGTGGGCGGAGGCCTGGTATCCCACCCTGTAGCCTCACCTTTCAAGGGACAGAATGGAGCAGTTCTCAGCCTAGGCACCACTGACGGCCTCCAAGGTGTGGTCATGGCCAGTGGGTTGGTTTATATGCTTGAAGCCTTCCTGATGCTAGACTAACCCTGGGCACTCTGCAGCCCCAGCTGCTGGGTCGCTGGCTGAGGGGTGCTGCTGCTTCCAGGAAGCATGGAGGGAGACCTCCCTGGGCTAGCCTAGGCTGACATAGGCTGACCACCCCTCTCTCATTCCATATGCTTAAGCAGAGGTCACAGAGAGGACCCCAGCCTGGTCTCGTCATGTGTGTAATGCAGACTGTTGTGTTTCTGATAAGGGCCTGGCCTCTCCCAATGGAGCAGTAGATTGGGAGTGTGAAGGACTCAGGTGCCCCATTCCCACGGCTGGAGCCAAGACCAGAAGCCCATGTCAGGGTCCAAGTGGAGTGGTGTGGTGTGTGGGAGGGGGCCCTGTCTTGGCAGGACACAGCCCACATCTCAACTTCCTGATGGCTGCTCCCTCATCCCATCCAGAGGCAAGGCACTCATGAAGCCTCAAAGACAGGTTTGGAAAGTGTTTTCACAGAAGTGTTTTGTCTCCTCCTCCAGTGTGATCCTTACATCAAGATCTCCATAGGGAAGAAATCAGTGAGTGACCAGGATAACTACATCCCCTGCACGCTGGAGCCCGTATTTGGAAA G TAAATTGGGGCATCTTGGGTCTTGGGGTGGAGGAGCCAGACAGGATAACCCACAGTCTAGTGGGGGAGATGTGACTGGCACTGTGAAGTCCGTATCTCTTGGAGCAAAACTGTATTCCTTAAATCTTGCATGTCTATGGGGGCATAGCCTCAGTTAGCCCTTCTTTAGCTGCTAAAGACTTGATCCCAACAGAAGCTCCTAGTTAAATGCTAAACAATTACGTCTAAGATCAGAAATCTACATGGCTTGAGCTCAGGAGTTTGAGACCAGTCTGGGCAACATAGCAAGACCCCATCTCTACAAAAAATAAAAAACTTAGCCAGGCATGGTAGTGCATGCCTGTGGTTCCAGATACTTGGGAGGCTGAGGTGGGAGGATCCCTTGAGCCTAGGAGTTTGAGCTTACAGTGATCTGTGATCAAACTACTGCACTCCAGCCTGGGTGACAGAGCAAAACCCTGTCTCAAAAAAAAAAAAAAAAAAAAAGAAAGAAAAAGTAGAAATCTGTGTGTAAGCTCAGAAGATGCATTTGCTAATTCTTTTTTTTTTTTAAGATAAACTTTTACTTTGGAATAATTTTAGATTTACTAAACAGTTACAGAGACAGTGCATAATTTCCACTTACTCAATTTCCCCCACTGGTAACCCCTTACTCAATTTCCCCCACTGGTATCATCTTACCTAACCATGGGACATTTGTCAAAATGAAGAAATCATCATTGGTGCATTACGTTTAACCAAACTAGACTTTTTTTTAAAGGATTTCATGTTTTCCCACTAATGTCCTCTTACTAATGTCTCAGGATCCAATCTGTTATACGACATTGCATTTAGTAATTAATTCTCTGTAAGTGTCCCACATGTGTTTGTAGCAAGAGCTAGAGGAAGAATGTAGACCCAAGATAGTAAAATGATTCCTGAAATCTGAGAACAAGGAAAGGAAGAATCAATGCCCATTGCATGGGAGTAATTCCTAGGCATCCTGAATTGCTGTTTGGATGTGAGCTTGTTTAGGCCAGAGAGGGGAGGATGCAGAGGGAGGGTGGCAGCTATTTCTCTCGCACCTGCAGAAGACACCTACTGTTCCGATGTCATCAAGTCACATATTGCACTTATAAACCGGGGTCTGTTGAGTTAGTTGTGGTCACTCACCCTGCAGTGAGGACCCAGTCTCCTTGGGGAAATGCAGCTTTTGAAGGTGAACCAGCCCTGGCCAGTGATGAGCTCCCACCAAGCAGGGATTGGAGGGAAGTGGTAGGTGGGAGAGAGGAACTCTGTATGAGTTCCAGGGGCTGGCGAGCTCCAGGGGCTGGCGCAGGACCTCAGGCTAATGCTGATGGGGCAGTGGGCAGGGCCTGTTCCACAGTGGGTGGGCGTGTGTCAGGACTCAGGGGAGATAGGGGAGCCAGTGCAGGAACACACCCCTCCCATGCCTGTTTCCCCATCTCCCCCCAATCCTGGGCAAGCTGGTCTGCCTGGTGCTTTAAGCCTCCTTCAGATCACAACAGAAACCCTTCCATTCTATAACCCATGTGTCAGACGAACACAACGCATGAGGTGGTTTTTGTCACCATGTATTCCATGGCGTTACCTTCTCCTTTCCCCTGAGGAAACTGACAGGCAGGTGACTTTTCTGCTCACATTGAGCTCAGCCACTTTAACTCACGAACCCAGTCTCAGGCCATAACCCACTGCCCATTTGTATGGAATCCCGAGCTCCCTGATGGCTCTAGGGGAGTGTGTCCCCTCTTTGGATGCAGTTGTGTGTGTATATGTGTGTATGTATGTGTGTGTATGTGTGTATATGTGCACATATGTGCGTGCGTGTGTATGTATTTGTGTGTATGTGTGTGTATATGTGTATGTGTGTCTGTGTTCGTGTGTCTGTGTGGTGTGTGTATATATGTGTGCATGTGTATTTGTGTGTATACATGTGTGTAGGTGTATGCGTCTGTGTGTGTCTGTGTGTCTGTGTGGCATGTGTGTATTTTGTGTGTATGTGTCTCTGTGTCCATGTGTCCGTGTATGTGTGTGGTGTGTGTATATGTATGTGTGTTGTATGTATGTGTGTGTATGTGTATTTGTATATGTGTGCGTGTATGTGCATGTGTATTTGTATGTGTGTGTGTATGTGTGTTTGTGTCTGTGTGTCTGTGTGTGGTGTGTGTATATGTGCATTTGTGTATATGTGTGTGTATGTGTGTGTGTCTGTGTCCATGTATGTGTGTGTGTGTACGTGAGTACATTTGTGTGCCTGTGTGTGTATATGTGTGCACGTATGTGCATGTGTGTGTGTGTGCATATTTGTGTGTGTATGTCTGTGTCCATGTGTCTGCATATGTGTGTGGTGTGTGTATATTTGTGTATGTTTACGTGTGTGTGTGTGTGTGCGCGCACGCGCGTGGTGTAGGGAAGGAAGGGCCCTGCCTTCCTCCCTTCCTGGTCCAGTGTTTCTCCCTTCCTGCTCTGGCTGACCTCTGAGGTTCTGACTCCTGCAGTGTCTGGGCTGGGGAGAGGGCCCGTCTCATGTGCCCACCACCGCTGTCATCCGGGTACTCTCTGGACCACGGATGTTGAAAGCCGACTTATTTTCCTGTGGGTGCTTTCCAGAGGTTCCTCAGAGAGCCCCCCGTGAGCCCTCCCACTGCACTTTCTGGGATATGGCAGATGCTGCGTCCCTTTAGCTGCTGTCCCCAGCCCCTGGTTCTCAGATGGTCAACTCCACAATCTCTCGCTGCTACAACCCTCTGGGGGTCTCACAGGGCAGGATTCAGAGCAGTTCCAGTCTGGCCTCAGAGTGGTTTCTTCTCCTCCCAGGAAGTCCTTGTGTTCCTTGCCCAGGCATGGGCCAGAGTGCAGCTCCTCCCAAATGTGGCCCCTGCCCTCCTTCTCCATGCCACAAGTTGCTTAAGTTTCCCTGAGCATGCACCAGGTGCCAGGCTTGTGTCTCTCACCTGCAGGGAGCTCATTTTAGGGGGAGAAGGGAGAATACCTCTCCCTTTGATGCACATGGTAGATGGAGTTCTCAGATACAGCAAGAGCTCTCGCCAGAGAAATCTTTTCACAAATTCTCCCTCCATCCCATCCCAGAAGTGGATGTGAAGAGTCCAGAAAGCAGGTCCCAGGCCTGGAGTTTCCAGTCATCGGTAAATTCCAAAGCTCTGGCCCCTTGCTCTGGAGTACCAGTCGTCGGTAAATTGGAGTCCCCCAATTTACCGGGAGTCCTCCCTGGACTGGAGGTACCAGTCGTCGGTAAATTCCAAAGCTCTGGCCCCTGGCTCTGGAGTTTCACATCTCTTGCATCTGTTGTCTCCTGGTGTCGTGGTCAAAGCTTCAGTTTTAATGTGCATTTCCAATTCATTCTTTCGGTCTGTGGTCCATCAGGCAGGCACTTGCCTTATGCCCAGCACAGTTTATTTGGGAAAGTCCTGTCCCCCTTCCCCCTACACACACACTCAGGCCCAGTACAGCAGTGCTGTGGGTGGTTGGGCCTGTAAGATCTGTAGGGGGCCCAAGGAAAGAAGACTCCCTGGGGTAGTTTCGAGCTCTTGTCCTGCCCTGCCTGTCCCTTGGGTGCCCCGTGTTGGCTGACATCGGGAATCTGCCCCTCCTGCAG G ATGTTCGAGCTGACCTGCACTCTGCCTCTGGAGAAGGACCTAAAGATCACTCTCTATGACTATGACCTCCTCTCCAAGGACGAAAAGATCGGTGAGACGGTCGTCGACCTGGAGAACAGGCTGCTGTCCAAGTTTGGGGCTCGCTGTGGACTCCCACAGACCTACTGTGTGTACGTGGATGGGGGCTGGCTGCCTGCTTCTCTGACAACACACCACCCCTGTCTTCTCTGACAACACACCACCACTGAGCACTTACTGTGTGCCAGCCCTGGGCTTAGCACTTCCTAGGCATTCTCTCATTGAGTCCAATGGGAGTCCTATCCCCACTCCACAGATGAAGAAACTGAAGCCCAGAGATGTTATTGCTTGTAAGTGGTGGAATTAGGATTTGAACCAAGAACCTGGCTCATCACATTGTTATAATCCAGTTATCTGTAATGCACATAGAAGGCCTAGAGAGGGCTAGGTACCTGGAAAGGGAGAGAGGGAAGGAAGGCAGGAAAGAAGCAGGGGAAACAGATGAGAGGACATGTGTGCTGCAACTGGGCCCGAAGGGGAATTTTGTGATGGTTTATGTCAGGGGAATGCATGTGAGGACTGCACCCCTCTTCCCACCATCTCAAGTCTTCTCTGGGTCTGATTATCTAACTCTGGAAATTGAAAACATTTAAGTTGCAATTCCGTACTTAAATGAGTCCTTTTCTCTCTGAGCCTCCATTTCTCCATCTGTAAAATGGGGATGCCCAGTCATGGTGAGCAATCAGATGGGACACCCACTGTAAAAGCAAGGAGTGGGCAATGCTGTACATGGGGGTACACCAGTCCCTGCATGCCCCTCTACCCTCATGAGTGTCCTTGAAGCATCTCATCTATGTCTTGTGCT TGCTCCTCAG>DYSF Intron 43i through intron 45i in the context of Homosapiens chromosome 2, alternate assembly CHM1_1.1, whole genomeshotgun sequence, NCBI Reference Sequence: NC_018913.2: SEQ ID NO: 118GTAACTTTCCTAGAGCCCTCACCTCCCCCAGAGTAGCAGGCTCAGGTACAAGTGGCCTATAGAACCTGGACACAAACTCTGCCTCAGGGAGTTCATAGTAGGTTGGGAAACAGACAAACACACAAAACTGAGAGGTGCCTGGATGGAGTTGTGTTAAGGACCAAGTGCTCTAGAAGGTCAGGGAAGGCCAGTGTCAGTACAGGACTTTGGGAAAATGGGGAAGGCTTCCTGGAAGGAATAGGACTCTTAGGATAGATGAGATTTTGATAGGCTGGGAGGGAGAAGAAAATAGTGTTTTAAGAGGGGCCAAAAGCATAGGCAAAGGTTTGAAAGAAAATTGCTTAATGTGTGTCTAGAACAAGAATAAAGCAGAAAGATAAGTAAGGCTGAGGGTATAGGTTGGGACTCTCAATGGTGAATGGGGAGGAGTGATGGGGGAAAGGAGCAGCGTTAGTGACTGCTATGCCTAGTCATGGCAGAGTCCCAGGTAAAAGGGAATGCTGGTAACACTCACCCGGTCTTTATTTAAAATTTTGATATTTTGTTCATCATGGACTATTGACACTGATTTTAATTTTTAAAATATTTCATTAAAGTACTGTTTATCTTAATAACTATTTTGGCCCTTCCTTACATTTTGTGCCCAAGATAAGTGCGTGAGCTGTCTCCCTCTAGTCATTGCCTGAGATGGAGAGGGGAGATTTGAAGGAGGAAAGGAGTCTGGAGTTATGGGGAGGTGACAGAGGTGGGAGGCAGAGTAATGTTGGGAAGAGAAAGAAAGAGGAAAGAAGAGAGGCAGGGGTGGGGCCTGATTTGCCTTTACAGGCATGAGGCTAGGGTCCCCTCTTGTCTCCTGAGGCAGGTTTAGGCCCAGCATGTGTCCTTCAGGTGGTGGGTGGAGGCCTGGTATCCCACCCTGTAGCCTCACCTTTCAAGGGACAGAATGGAGCAGTTCTCAGCCTAGGCACCACTGACGGCCTCCAAGGTGTGGTCATGGCCAGTGGGTTGGTTTATATGCTTGAAGCCTTCCTGATGCTAGACTAACCCTGGGCACTCTGCAGCCCCAGCTGCTGGGTCGCTGGCTGAGGGGTGCTGCTGCTTCCAGGAAGCATGGAGGGAGACCTCCCTGGGCTAGCCTAGGCTGACATAGGCTGACCACCCCTCTCTCATTCCATATGCTTAAGCAGAGGTCACAGAGAGGACCCCAGCCTGGTCTCGTCATGTGTGTAATGCAGACTGTTGTGTTTCTGATAAGGGCCTGGCCTCTCCCAATGGAGCAGTAGATTGGGAGTGTGAAGGACTCAGGTGCCCCATTCCCACGGCTGGAGCCAAGACCAGAAGCCCATGTCAGGGTCCAAGTGGAGTGGTGTGGTGTGTGGGAGGGGGCCCTGTCTTGGCAGGACACAGCCCACATCTCAACTTCCTGATGGCTGCTCCCTCATCCCATCCAGAGGCAAGGCACTCATGAAGCCTCAAAGACAGGTTTGGAAAGTGTTTTCACAGAAGTGTTTTGTCTCCTCCTCCAGTGTGATCCTTACATCAAGATCTCCATAGGGAAGAAATCAGTGAGTGACCAGGATAACTACATCCCCTGCACGCTGGAGCCCGTATTTGGAAAGTAAATTGGGGCATCTTGGGTCTTGGGGTGGAGGAGCCAGACAGGATAACCCACAGTCTAGTGGGGGAGATGTGACTGGCACTGTGAAGTCCGTATCTCTTGGAGCAAAACTGTATTCCTTAAATCTTGCATGTCTATGGGGGCATAGCCTCAGTTAGCCCTTCTTTAGCTGCTAAAGACTTGATCCCAACAGAAGCTCCTAGTTAAATGCTAAACAATTACGTCTAAGATCAGAAATCTACATGGCTTGAGCTCAGGAGTTTGAGACCAGTCTGGGCAACATAGCAAGACCCCATCTCTACAAAAAATAAAAAACTTAGCCAGGCATGGTAGTGCATGCCTGTGGTTCCAGATACTTGGGAGGCTGAGGTGGGAGGATCCCTTGAGCCTAGGAGTTTGAGCTTACAGTGATCTGTGATCAAACTACTGCACTCCAGCCTGGGTGACAGAGCAAAACCCTGTCTCAAAAAAAAAAAAAAAAAAAAAGAAAGAAAAAGTAGAAATCTGTGTGTAAGCTCAGAAGATGCATTTGCTAATTCTTTTTTTTTTTTAAGATAAACTTTTACTTTGGAATAATTTTAGATTTACTAAACAGTTACAGAGACAGTGCATAATTTCCACTTACTCAATTTCCCCCACTGGTAACCCCTTACTCAATTTCCCCCACTGGTATCATCTTACCTAACCATGGGACATTTGTCAAAATGAAGAAATCATCATTGGTGCATTACGTTTAACCAAACTAGACTTTTTTTTAAAGGATTTCATGTTTTCCCACTAATGTCCTCTTACTAATGTCTCAGGATCCAATCTGTTATACGACATTGCATTTAGTAATTAATTCTCTGTAAGTGTCCCACATGTGTTTGTAGCAAGAGCTAGAGGAAGAATGTAGACCCAAGATAGTAAAATGATTCCTGAAATCTGAGAACAAGGAAAGGAAGAATCAATGCCCATTGCATGGGAGTAATTCCTAGGCATCCTGAATTGCTGTTTGGATGTGAGCTTGTTTAGGCCAGAGAGGGGAGGATGCAGAGGGAGGGTGGCAGCTATTTCTCTCGCACCTGCAGAAGACACCTACTGTTCCGATGTCATCAAGTCACATATTGCACTTATAAACCGGGGTCTGTTGAGTTAGTTGTGGTCACTCACCCTGCAGTGAGGACCCAGTCTCCTTGGGGAAATGCAGCTTTTGAAGGTGAACCAGCCCTGGCCAGTGATGAGCTCCCACCAAGCAGGGATTGGAGGGAAGTGGTAGGTGGGAGAGAGGAACTCTGTATGAGTTCCAGGGGCTGGCGAGCTCCAGGGGCTGGCGCAGGGCCTCAGGCTAATGCTGATGGGGCAGTGGGCAGGGCCTGTTCCACAGTGGGTGGGGGTGTGTCAGGACTCAGGGGAGATAGGGGAGCCAGTGCAGGAACACACCCCTCCCATGCCTGTTTCCCCATCTCCCCCCAATCCTGGGCAAGCTGGTCTGCCTGGTGCTTTAAGCCTCCTTCAGATCACAACAGAAACCCTTCCATTCTATAACCCATGTGTCAGACGAACACAACGCATGAGGTGGTTTTTGTCACCATGTATTCCATGGCGTTACCTTCTCCTTTCCCCTGAGGAAACTGACAGGCAGGTGACTTTTCTGCTCACATTGAGCTCAGCCACTTTAACTCACGAACCCAGTCTCAGGCCATAACCCACTGCCCATTTGTATGGAATCCCGAGCTCCCTGATGGCTCTAGGGGAGTGTGTCCCCTCTTTGGATGCAGTTGTGTGTGTATATGTGTGTATGTATGTGTGTGTATGTGTGTATATGTGCACATATGTGCGTGCGTGTGTATGTATTTGTGTGTATGTGTGTGTATATGTGTATGTGTGTCTGTGTTCGTGTGTCTGTGTGGTGTGTGTATATATGTGTGCATGTGTATTTGTGTGTATACATGTGTGTAGGTGTATGCGTCTGTGTGTGTCTGTGTGTCTGTGTGGCATGTGTGTATTTTGTGTGTGTGTATGTGTCTCTGTGTCCATGTGTCCGTGTATGTGTGTGGTGTGTGTATATGTATGTGTGTTGTATGTATGTGTGTGTATGTGTATTTGTATATGTGTGCGTGTATGTGCATGTGTATTTGTATGTGTGTGTGTATGTGTGTTTGTGTCTGTGTGTCTGTGTGTGGTGTGTGTATATGTGCATTTGTGTATATGTGTGTGTATGTGTGTGTGTCTGTGTCCATGTATGTGTGTGTGTGTACGTGAGTACATTTGTGTGCCTGTGTGTGTATATGTGTGCACGTATGTGCATGTGTGTGTGTGTGCATATTTGTGTGTGTATGTCTGTGTCCATGTGTCTGCATATGTGTGTGGTGTGTGTATATTTGTGTATGTTTACGTGTGTGTGTGTGTGTGCGCGCACGCGCGTGGTGTAGGGAAGGAGGGGCCCTGCCTTCCTCCCTTCCTGGTCCAGTGTTTCTCCCTTCCTGCTCTGGCTGACCTCTGAGGTTCTGACTCCTGCAGTGTCTGGGCTGGGGAGAGGGCCCGTCTCATGTGCCGACCACCGCTGTCATCCGGGTACTCTCTGGACCACGGATGTTGAAAGCCGACTTATTTTCCTGTGGGTGCTTTCCAGAGGTTCCTCAGAGAGCCCCCCGTGAGCCCTCCCACTGCACTTTCTGGGATATGGCAGATGCTGCGTCCCTTTAGCTGCTGTCCCCAGCCCCTGGTTCTCAGATGGTCAACTCCACAATCTCTCGCTGCTACAACCCTCTGGGGGTCTCACAGGGCAGGATTCAGAGCAGTTCCAGTCTGGCCTCAGAGTGGTTTCTTCTCCTCCCAGGAAGTCCTTGTGTTCCTTGCCCAGGCATGGGCCAGAGTGCAGCTCCTCCCAAATGTGGCCCCTGCCCTCCTTCTCCATGCCACAAGTTGCTTAAGTTTCCCTGAGCATGCACCAGGTGCCAGGCTTGTGTCTCTCACCTGCAGGGAGCTCATTTTAGGGGGAGAAGGGAGAATACCTCTCCCTTTGATGCACATGGTAGATGGAGTTCTCAGATACAGCAAGAGCTCTCGCCAGAGAAATCTTTTCACAAATTCTCCCTCCATCCCATCCCAGAAGTGGATGTGAAGAGTCCAGAAAGCAGGTCCCAGGCCTGGAGTTTCCAGTCATCGGTAAATTCCAAAGCTCTGGCCCCTTGCTCTGGAGTACCAGTCGTCGGTAAATTGGAGTCCCCCAATTTACCGGGAGTCCTCCCTGGACTGGAGGTACCAGTCGTCGGTAAATTCCAAAGCTCTGGCCCCTGGCTCTGGAGTTTCACATCTCTTGCATCTGTTGTCTCCTGGTGTCGTGGTCAAAGCTTCAGTTTTAATGTGCATTTCCAATTCATTCTTTCGGTCTGTGGTCCATCAGGCAGGCACTTGCCTTATGCCCAGCACAGTTTATTTGGGAAAGTCCTGTCCCCCTTCCCCCTACACACACATTCAGGCCCAGTACAGCAGTGCTGTGGGTGGTTGGGCCTGTAAGATCTGTAGGGGGCCCAAGGAAAGAAGACTCCCTGGGGTAGTTTCGAGCTCTTGTCCTGCCCTGCCTGTCCCTTGGGTGCCCTGTGTTGGCTGACATCGGGAATCTGCCCCTCCTGCAGGATGTTCGAGCTGACCTGCACTCTGCCTCTGGAGAAGGACCTAAAGATCACTCTCTATGACTATGACCTCCTCTCCAAGGACGAAAAGATCGGTGAGACGGTCGTCGACCTGGAGAACAGGCTGCTGTCCAAGTTTGGGGCTCGCTGTGGACTCCCACAGACCTACTGTGTGTACGTGGATGGGGGCTGGCTGCCTGCTTCTCTGACAACACACCACCCCTGTCTTCTCTGACAACACACCACCACTGAGCACTTACTGTGTGCCAGCCCTGGGCTTAGCACTTCCTAGGCATTCTCTCATTGAGTCCAATGGGAGTCCTATCCCCACTCCACAGATGAAGAAACTGAAGCCCAGAGATGTTATTGCTTGTAAGTGGTGGAATTAGGATTTGAACCAAGAACCTGGCTCATCACATTGTTATAATCCAGTTATCTGTAATGCACATAGAAGGCCTAGAGAGGGCTAGGTACCTGGAAAGGGAGAGAGGGAAGGAAGGCAGGAAAGAAGCAGGGGAAACAGATGAGAGGACATGTGTGCTGCAACTGGGCCCGAAGGGGAATTTTGTGATGGTTTATGTCAGGGGAATGCATGTGAGGACTGCACCCCTCTTCCCACCATCTCAAGTCTTCTCTGGGTCTGATTATCTAACTCTGGAAATTGAAAACATTTAAGTTGCAATTCCGTACTTAAATGAGTCCTTTTCTCTCTGAGCCTCCATTTCTCCATCTGTAAAATGGGGATGCCCAGTCATGGTGAGCAATCAGATGGGACACCCACTGTAAAAGCAAGGAGTGGGCAATGCTGTACATGGGGGTACACCAGTCCCTGCATGCCCCTCTACCCTCATGAGTGTCCTTGAAGCATCTCATCTATGTCTTGTGCTTGCTCCTCAG>DYSF Intron 43i through intron 45i in the context of Homosapiens chromosome 2, alternate assembly HuRef whole genomeshotgun sequence, NCBI Reference Sequence: AC_000134.1: SEQ ID NO: 119GTAACTTTCCTAGAGCCCTCACCTCCCCCAGAGTAGCAGGCTCAGGTACAAGTGGCCTATAGAACCTGGACACAAACTCTGCCTCAGGGAGTTCATAGTAGGTTGGGAAACAGACAAACACACAAAACTGAGAGGTGCCTGGATGGAGTTGTGTTAAGGACCAAGTGCTCTAGAAGGTCAGGGAAGGCCAGTGTCAGTACAGGACTTTGGGAAAATGGGGAAGGCTTCCTGGAAGGAATAGGACTCTTAGGATAGATGAGATTTTGATAGGCTGGGAGGGAGAAGAAAATAGTGTTTTAAGAGGGGCCAAAAGCATAGGCAAAGGTTTGAAAGAAAATTGCTTAATGTGTGTCTAGAACAAGAATAAAGCAGAAAGATAAGTAAGGCTGAGGGTATAGGTTGGGACTCTCAATGGTGAATGGGGAGGAGTGATGGGGGAAAGGAGCAGCGTTAGTGACTGCTATGCCTAGTCATGGCAGAGTCCCAGGTAAAAGGGAATGCTGGTAACACTCACCCGGTCTTTATTTAAAATTTTGATATTTTGTTCATCATGGACTATTGACACTGATTTTAATTTTTAAAATATTTCATTAAAGTACTGTTTATCTTAATAACTATTTTGGCCCTTCCTTACATTTTGTGCCCAAGATAAGTGCGTGAGCTGTCTCCCTCTAGTCATTGCCTGAGATGGAGAGGGGAGATTTGAAGGAGGAAAGGAGTCTGGAGTTATGGGGAGGTGACAGAGGTGGGAGGCAGAGTAATGTTGGGAAGAGAAAGAAAGAGGAAAGAAGAGAGGCAGGGGTGGGGCCTGATTTGCCTTTACAGGCATGAGGCTAGGGTCCCCTCTTGTCTCCTGAGGCAGGTTTAGGCCCAGCATGTGTCCTTCAGGTGGTGGGTGGAGGCCTGGTATCCCACCCTGTAGCCTCACCTTTCAAGGGACAGAATGGAGCAGTTCTCAGCCTAGGCACCACTGACGGCCTCCAAGGTGTGGTCATGGCCAGTGGGTTGGTTTATATGCTTGAAGCCTTCCTGATGCTAGACTAACCCTGGGCACTCTGCAGCCCCAGCTGCTGGGTCGCTGGCTGAGGGGTGCTGCTGCTTCCAGGAAGCATGGAGGGAGACCTCCCTGGGCTAGCCTAGGCTGACATAGGCTGACCACCCCTCTCTCATTCCATATGCTTAAGCAGAGGTCACAGAGAGGACCCCAGCCTGGTCTCGTCATGTGTGTAATGCAGACTGTTGTGTTTCTGATAAGGGCCTGGCCTCTCCCAATGGAGCAGTAGATTGGGAGTGTGAAGGACTCAGGTGCCCCATTCCCACGGCTGGAGCCAAGACCAGAAGCCCATGTCAGGGTCCAAGTGGAGTGGTGTGGTGTGTGGGAGGGGGCCCTGTCTTGGCAGGACACAGCCCACATCTCAACTTCCTGATGGCTGCTCCCTCATCCCATCCAGAGGCAAGGCACTCATGAAGCCTCAAAGACAGGTTTGGAAAGTGTTTTCACAGAAGTGTTTTGTCTCCTCCTCCAGTGTGATCCTTACATCAAGATCTCCATAGGGAAGAAATCAGTGAGTGACCAGGATAACTACATCCCCTGCACGCTGGAGCCCGTATTTGGAAAGTAAATTGGGGCATCTTGGGTCTTGGGGTGGAGGAGCCAGACAGGATAACCCACAGTCTAGTGGGGGAGATGTGACTGGCACTGTGAAGTCCGTATCTCTTGGAGCAAAACTGTATTCCTTAAATCTTGCATGTCTATGGGGGCATAGCCTCAGTTAGCCCTTCTTTAGCTGCTAAAGACTTGATCCCAACAGAAGCTCCTAGTTAAATGCTAAACAATTACGTCTAAGATCAGAAATCTACATGGCTTGAGCTCAGGAGTTTGAGACCAGTCTGGGCAACATAGCAAGACCCCATCTCTACAAAAAATAAAAAACTTAGCCAGGCATGGTAGTGCATGCCTGTGGTTCCAGATACTTGGGAGGCTGAGGTGGGAGGATCCCTTGAGCCTAGGAGTTTGAGCTTACAGTGATCTGTGATCAAACTACTGCACTCCAGCCTGGGTGACAGAGCAAAACCCTGTCTCAAAAAAAAAAAAAAAAAAAAAGAAAGAAAAAGTAGAAATCTGTGTGTAAGCTCAGAAGATGCATTTGCTAATTCTTTTTTTTTTTTAAGATAAACTTTTACTTTGGAATAATTTTAGATTTACTAAACAGTTACAGAGACAGTGCATAATTTCCACTTACTCAATTTCCCCCACTGGTAACCCCTTACTCAATTTCCCCCACTGGTATCATCTTACCTAACCATGGGACATTTGTCAAAATGAAGAAATCATCATTGGTGCATTACGTTTAACCAAACTAGACTTTTTTTTAAAGGATTTCATGTTTTCCCACTAATGTCCTCTTACTAATGTCTCAGGATCCAATCTGTTATACGACATTGCATTTAGTAATTAATTCTCTGTAAGTGTCCCACATGTGTTTGTAGCAAGAGCTAGAGGAAGAATGTAGACCCAAGATAGTAAAATGATTCCTGAAATCTGAGAACAAGGAAAGGAAGAATCAATGCCCATTGCATGGGAGTAATTCCTAGGCATCCTGAATTGCTGTTTGGATGTGAGCTTGTTTAGGCCAGAGAGGGGAGGATGCAGAGGGAGGGTGGCAGCTATTTCTCTCGCACCTGCAGAAGACACCTACTGTTCCGATGTCATCAAGTCACATATTGCACTTATAAACCGGGGTCTGTTGAGTTAGTTGTGGTCACTCACCCTGCAGTGAGGACCCAGTCTCCTTGGGGAAATGCAGCTTTTGAAGGTGAACCAGCCCTGGCCAGTGATGAGCTCCCACCAAGCAGGGATTGGAGGGAAGTGGTAGGTGGGAGAGAGGAACTCTGTATGAGTTCCAGGGGCTGGCGAGCTCCAGGGGCTGGCGCAGGGCCTCAGGCTAATGCTGATGGGGCAGTGGGCAGGGCCTGTTCCACAGTGGGTGGGGGTGTGTCAGGACTCAGGGGAGATAGGGGAGCCAGTGCAGGAACACACCCCTCCCATGCCTGTTTCCCCATCTCCCCCCAATCCTGGGCAAGCTGGTCTGCCTGGTGCTTTAAGCCTCCTTCAGATCACAACAGAAACCCTTCCATTCTATAACCCATGTGTCAGACGAACACAACGCATGAGGTGGTTTTTGTCACCATGTATTCCATGGCGTTACCTTCTCCTTTCCCCTGAGGAAACTGACAGGCAGGTGACTTTTCTGCTCACATTGAGCTCAGCCACTTTAACTCACGAACCCAGTCTCAGGCCATAACCCACTGCCCATTTGTATGGAATCCCGAGCTCCCTGATGGCTCTAGGGGAGTGTGTCCCCTCTTTGGATGCAGTTGTGTGTGTATATGTGTGTATGTATGTGTGTGTATGTGTGTATATGTGCACATATGTGCGTGCGTGTGTATGTATTTGTGTGTGTGTGTGTATATGTGTATGTGTGTGTTCGTGTGTCTGTGTGGTGTGTGTATATATGTGTGCATGTGTATTTGTGTGTATACATGTGTGTAGGTGTATGCGTCTGTGTGTGTCTGTGTGTCTGTGTGGCATGTGTGTATTTTGTGTGTGTGTATGTGTCTCTGTGTCCATGTGTCCGTGTATGTGTGTGGTGTGTGTATATGTATGTGTGTTGTATGTATGTGTGTGTATGTGTATTTGTATATGTGTGCGTGTATGTGCATGTGTATTTGTATGTGTGTGTGTATGTGTGTTTGTGTCTGTGTGTCTGTGTGTGGTGTGTGTATATGTGCATTTGTGTATATGTGTGTGTATGTGTGTGTGTCTGTGTCCATGTATGTGTGTGTGTGTACGTGAGTACATTTGTGTGCCTGTGTGTGTATATGTGTGCACGTATGTGCATGTGTGTGTGTGTGCATATTTGTGTGTGTATGTCTGTGTCCATGTGTCTGCATATGTGTGTGGTGTGTGTATATTTGTGTATGTTTACGTGTGTGTGTGTGTGTGCGCGCACGCGCGTGGTGTAGGGAAGGAAGGGCCCTGCCTTCCTCCCTTCCTGGTCCAGTGTTTCTCCCTTCCTGCTCTGGCTGACCTCTGAGGTTCTGACTCCTGCAGTGTCTGGGCTGGGGAGAGGGCCCGTCTCATGTGCCCACCACCGCTGTCATCCGGGTACTCTCTGGACCACGGATGTTGAAAGCCGACTTATTTTCCTGTGGGTGCTTTCCAGAGGTTCCTCAGAGAGCCCCCCGTGAGCCCTCCCACTGCACTTTCTGGGATATGGCAGATGCTGCGTCCCTTTAGCTGCTGTCCCCAGCCCCTGGTTCTCAGATGGTCAACTCCACAATCTCTCGCTGCTACAACCCTCTGGGGGTCTCACAGGGCAGGATTCAGAGCAGTTCCAGTCTGGCCTCAGAGTGGTTTCTTCTCCTCCCAGGAAGTCCTTGTGTTCCTTGCCCAGGCATGGGCCAGAGTGCAGCTCCTCCCAAATGTGGCCCCTGCCCTCCTTCTCCATGCCACAAGTTGCTTAAGTTTCCCTGAGCATGCACCAGGTGCCAGGCTTGTGTCTCTCACCTGCAGGGAGCTCATTTTAGGGGGAGAAGGGAGAATACCTCTCCCTTTGATGCACATGGTAGATGGAGTTCTCAGATACAGCAAGAGCTCTCGCCAGAGAAATCTTTTCACAAATTCTCCCTCCATCCCATCCCAGAAGTGGATGTGAAGAGTCCAGAAAGCAGGTCCCAGGCCTGGAGTTTCCAGTCATCGGTAAATTCCAAAGCTCTGGCCCCTTGCTCTGGAGTACCAGTCGTCGGTAAATTGGAGTCCCCCAATTTACCGGGAGTCCTCCCTGGACTGGAGGTACCAGTCGTCGGTAAATTCCAAAGCTCTGGCCCCTGGCTCTGGAGTTTCACATCTCTTGCATCTGTTGTCTCCTGGTGTCGTGGTCAAAGCTTCAGTTTTAATGTGCATTTCCAATTCATTCTTTCGGTCTGTGGTCCATCAGGCAGGCACTTGCCTTATGCCCAGCACAGTTTATTTGGGAAAGTCCTGTCCCCCTTCCCCCTACACACACATTCAGGCCCAGTACAGCAGTGCTGTGGGTGGTTGGGCCTGTAAGATCTGTAGGGGGCCCAAGGAAAGAAGACTCCCTGGGGTAGTTTCGAGCTCTTGTCCTGCCCTGCCTGTCCCTTGGGTGCCCTGTGTTGGCTGACATCGGGAATCTGCCCCTCCTGCAGGATGTTCGAGCTGACCTGCACTCTGCCTCTGGAGAAGGACCTAAAGATCACTCTCTATGACTATGACCTCCTCTCCAAGGACGAAAAGATCGGTGAGACGGTCGTCGACCTGGAGAACAGGCTGCTGTCCAAGTTTGGGGCTCGCTGTGGACTCCCACAGACCTACTGTGTGTACGTGGATGGGGGCTGGCTGCCTGCTTCTCTGACAACACACCACCCCTGTCTTCTCTGACAACACACCACCACTGAGCACTTACTGTGTGCCAGCCCTGGGCTTAGCACTTCCTAGGCATTCTCTCATTGAGTCCAATGGGAGTCCTATCCCCACTCCACAGATGAAGAAACTGAAGCCCAGAGATGTTATTGCTTGTAAGTGGTGGAATTAGGATTTGAACCAAGAACCTGGCTCATCACATTGTTATAATCCAGTTATCTGTAATGCACATAGAAGGCCTAGAGAGGGCTAGGTACCTGGAAAGGGAGAGAGGGAAGGAAGGCAGGAAAGAAGCAGGGGAAACAGATGAGAGGACATGTGTGCTGCAACTGGGCCCGAAGGGGAATTTTGTGATGGTTTATGTCAGGGGAATGCATGTGAGGACTGCACCCCTCTTCCCACCATCTCAAGTCTTCTCTGGGTCTGATTATCTAACTCTGGAAATTGAAAACATTTAAGTTGCAATTCCGTACTTAAATGAGTCCTTTTCTCTCTGAGCCTCCATTTCTCCATCTGTAAAATGGGGATGCCCAGTCATGGTGAGCAATCAGATGGGACACCCACTGTAAAAGCAAGGAGTGGGCAATGCTGTACATGGGGGTACACCAGTCCCTGCATGCCCCTCTACCCTCATGAGTGTCCTTGAAGCATCTCATCTATGTCTTGTGCTTGCTCCTCAG > DYSF Intron 44 in the context of a c.4886 + 1249 (G > T)mutation: SEQ ID NO: 120GTAAATTGGGGCATCTTGGGTCTTGGGGTGGAGGAGCCAGACAGGATAACCCACAGTCTAGTGGGGGAGATGTGACTGGCACTGTGAAGTCCGTATCTCTTGGAGCAAAACTGTATTCCTTAAATCTTGCATGTCTATGGGGGCATAGCCTCAGTTAGCCCTTCTTTAGCTGCTAAAGACTTGATCCCAACAGAAGCTCCTAGTTAAATGCTAAACAATTACGTCTAAGATCAGAAATCTACATGGCTTGAGCTCAGGAGTTTGAGACCAGTCTGGGCAACATAGCAAGACCCCATCTCTACAAAAAATAAAAAACTTAGCCAGGCATGGTAGTGCATGCCTGTGGTTCCAGATACTTGGGAGGCTGAGGTGGGAGGATCCCTTGAGCCTAGGAGTTTGAGCTTACAGTGATCTGTGATCAAACTACTGCACTCCAGCCTGGGTGACAGAGCAAAACCCTGTCTCAAAAAAAAAAAAAAAAAAAAAGAAAGAAAAAGTAGAAATCTGTGTGTAAGCTCAGAAGATGCATTTGCTAATTCTTTTTTTTTTTTAAGATAAACTTTTACTTTGGAATAATTTTAGATTTACTAAACAGTTACAGAGACAGTGCATAATTTCCACTTACTCAATTTCCCCCACTGGTAACCCCTTACTCAATTTCCCCCACTGGTATCATCTTACCTAACCATGGGACATTTGTCAAAATGAAGAAATCATCATTGGTGCATTACGTTTAACCAAACTAGACTTTTTTTTAAAGGATTTCATGTTTTCCCACTAATGTCCTCTTACTAATGTCTCAGGATCCAATCTGTTATACGACATTGCATTTAGTAATTAATTCTCTGTAAGTGTCCCACATGTGTTTGTAGCAAGAGCTAGAGGAAGAATGTAGACCCAAGATAGTAAAATGATTCCTGAAATCTGAGAACAAGGAAAGGAAGAATCAATGCCCATTGCATGGGAGTAATTCCTAGGCATCCTGAATTGCTGTTTGGATGTGAGCTTGTTTAGGCCAGAGAGGGGAGGATGCAGAGGGAGGGTGGCAGCTATTTCTCTCGCACCTGCAG >Intron 44: SEQ ID NO: 121GTAAATTGGGGCATCTTGGGTCTTGGGGTGGAGGAGCCAGACAGGATAACCCACAGTCTAGTGGGGGAGATGTGACTGGCACTGTGAAGTCCGTATCTCTTGGAGCAAAACTGTATTCCTTAAATCTTGCATGTCTATGGGGGCATAGCCTCAGTTAGCCCTTCTTTAGCTGCTAAAGACTTGATCCCAACAGAAGCTCCTAGTTAAATGCTAAACAATTACGTCTAAGATCAGAAATCTACATGGCTTGAGCTCAGGAGTTTGAGACCAGTCTGGGCAACATAGCAAGACCCCATCTCTACAAAAAATAAAAAACTTAGCCAGGCATGGTAGTGCATGCCTGTGGTTCCAGATACTTGGGAGGCTGAGGTGGGAGGATCCCTTGAGCCTAGGAGTTTGAGCTTACAGTGATCTGTGATCAAACTACTGCACTCCAGCCTGGGTGACAGAGCAAAACCCTGTCTCAAAAAAAAAAAAAAAAAAAAAGAAAGAAAAAGTAGAAATCTGTGTGTAAGCTCAGAAGATGCATTTGCTAATTCTTTTTTTTTTTTAAGATAAACTTTTACTTTGGAATAATTTTAGATTTACTAAACAGTTACAGAGACAGTGCATAATTTCCACTTACTCAATTTCCCCCACTGGTAACCCCTTACTCAATTTCCCCCACTGGTATCATCTTACCTAACCATGGGACATTTGTCAAAATGAAGAAATCATCATTGGTGCATTACGTTTAACCAAACTAGACTTTTTTTTAAAGGATTTCATGTTTTCCCACTAATGTCCTCTTACTAATGTCTCAGGATCCAATCTGTTATACGACATTGCATTTAGTAATTAATTCTCTGTAAGTGTCCCACATGTGTTTGTAGCAAGAGCTAGAGGAAGAATGTAGACCCAAGATAGTAAAATGATTCCTGAAATCTGAGAACAAGGAAAGGAAGAATCAATGCCCATTGCATGGGAGTAATTCCTAGGCATCCTGAATTGCTGTTTGGATGTGAGCTTGTTTAGGCCAGAGAGGGGAGGATGCAGAGGGAGGGTGGCAGCTATTTCTCTCGCACCTGCAGAAGACACCTACTGTTCCGATGTCATCAAGTCACATATTGCACTTATAAACCGGGGTCTGTTGAGTTAGTTGTGGTCACTCACCCTGCAGTGAGGACCCAGTCTCCTTGGGGAAATGCAGCTTTTGAAGGTGAACCAGCCCTGGCCAGTGATGAGCTCCCACCAAGCAGGGATTGGAGGGAAGTGGTAGGTGGGAGAGAGGAACTCTGTATGAGTTCCAGGGGCTGGCGAGCTCCAGGGGCTGGCGCAGGACCTCAGGCTAATGCTGATGGGGCAGTGGGCAGGGCCTGTTCCACAGTGGGTGGGCGTGTGTCAGGACTCAGGGGAGATAGGGGAGCCAGTGCAGGAACACACCCCTCCCATGCCTGTTTCCCCATCTCCCCCCAATCCTGGGCAAGCTGGTCTGCCTGGTGCTTTAAGCCTCCTTCAGATCACAACAGAAACCCTTCCATTCTATAACCCATGTGTCAGACGAACACAACGCATGAGGTGGTTTTTGTCACCATGTATTCCATGGCGTTACCTTCTCCTTTCCCCTGAGGAAACTGACAGGCAGGTGACTTTTCTGCTCACATTGAGCTCAGCCACTTTAACTCACGAACCCAGTCTCAGGCCATAACCCACTGCCCATTTGTATGGAATCCCGAGCTCCCTGATGGCTCTAGGGGAGTGTGTCCCCTCTTTGGATGCAGTTGTGTGTGTATATGTGTGTATGTATGTGTGTGTATGTGTGTATATGTGCACATATGTGCGTGCGTGTGTATGTATTTGTGTGTATGTGTGTGTATATGTGTATGTGTGTCTGTGTTCGTGTGTCTGTGTGGTGTGTGTATATATGTGTGCATGTGTATTTGTGTGTATACATGTGTGTAGGTGTATGCGTCTGTGTGTGTCTGTGTGTCTGTGTGGCATGTGTGTATTTTGTGTGTATGTGTCTCTGTGTCCATGTGTCCGTGTATGTGTGTGGTGTGTGTATATGTATGTGTGTTGTATGTATGTGTGTGTATGTGTATTTGTATATGTGTGCGTGTATGTGCATGTGTATTTGTATGTGTGTGTGTATGTGTGTTTGTGTCTGTGTGTCTGTGTGTGGTGTGTGTATATGTGCATTTGTGTATATGTGTGTGTATGTGTGTGTGTCTGTGTCCATGTATGTGTGTGTGTGTACGTGAGTACATTTGTGTGCCTGTGTGTGTATATGTGTGCACGTATGTGCATGTGTGTGTGTGTGCATATTTGTGTGTGTATGTCTGTGTCCATGTGTCTGCATATGTGTGTGGTGTGTGTATATTTGTGTATGTTTACGTGTGTGTGTGTGTGTGCGCGCACGCGCGTGGTGTAGGGAAGGAAGGGCCCTGCCTTCCTCCCTTCCTGGTCCAGTGTTTCTCCCTTCCTGCTCTGGCTGACCTCTGAGGTTCTGACTCCTGCAGTGTCTGGGCTGGGGAGAGGGCCCGTCTCATGTGCCCACCACCGCTGTCATCCGGGTACTCTCTGGACCACGGATGTTGAAAGCCGACTTATTTTCCTGTGGGTGCTTTCCAGAGGTTCCTCAGAGAGCCCCCCGTGAGCCCTCCCACTGCACTTTCTGGGATATGGCAGATGCTGCGTCCCTTTAGCTGCTGTCCCCAGCCCCTGGTTCTCAGATGGTCAACTCCACAATCTCTCGCTGCTACAACCCTCTGGGGGTCTCACAGGGCAGGATTCAGAGCAGTTCCAGTCTGGCCTCAGAGTGGTTTCTTCTCCTCCCAGGAAGTCCTTGTGTTCCTTGCCCAGGCATGGGCCAGAGTGCAGCTCCTCCCAAATGTGGCCCCTGCCCTCCTTCTCCATGCCACAAGTTGCTTAAGTTTCCCTGAGCATGCACCAGGTGCCAGGCTTGTGTCTCTCACCTGCAGGGAGCTCATTTTAGGGGGAGAAGGGAGAATACCTCTCCCTTTGATGCACATGGTAGATGGAGTTCTCAGATACAGCAAGAGCTCTCGCCAGAGAAATCTTTTCACAAATTCTCCCTCCATCCCATCCCAGAAGTGGATGTGAAGAGTCCAGAAAGCAGGTCCCAGGCCTGGAGTTTCCAGTCATCGGTAAATTCCAAAGCTCTGGCCCCTTGCTCTGGAGTACCAGTCGTCGGTAAATTGGAGTCCCCCAATTTACCGGGAGTCCTCCCTGGACTGGAGGTACCAGTCGTCGGTAAATTCCAAAGCTCTGGCCCCTGGCTCTGGAGTTTCACATCTCTTGCATCTGTTGTCTCCTGGTGTCGTGGTCAAAGCTTCAGTTTTAATGTGCATTTCCAATTCATTCTTTCGGTCTGTGGTCCATCAGGCAGGCACTTGCCTTATGCCCAGCACAGTTTATTTGGGAAAGTCCTGTCCCCCTTCCCCCTACACACACACTCAGGCCCAGTACAGCAGTGCTGTGGGTGGTTGGGCCTGTAAGATCTGTAGGGGGCCCAAGGAAAGAAGACTCCCTGGGGTAGTTTCGAGCTCTTGTCCTGCCCTGCCTGTCCCTTGGGTGCCCCGTGTTGGCTGACATCGGGAATCTGCCCCTCCTGCAG >Immunogenic sequence for anti-PE44.1 antibody: SEQ ID NO: 122CAFEGEPALASDELPPSRDWR

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,and/or methods, if such features, systems, articles, materials, and/ormethods are not mutually inconsistent, is included within the scope ofthe present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

1. A method of modulating splicing in a cell that contains a DYSF genecomprising a c.4886+1249 (G>T) mutation, the method comprising:delivering to the cell an antisense nucleic acid comprising 15 to 25nucleotides in length that targets a pre-messenger RNA expressed fromthe DYSF gene and alters splicing of the pre-messenger RNA such thatexons 44 and 45 of the pre-messenger RNA are spliced together without anintervening pseudoexon, wherein the antisense nucleic acid iscomplementary to a sequence as set forth in SEQ ID NO: 56 or SEQ ID NO:58.
 2. The method of claim 1, wherein the cell is heterozygous for thec.4886+1249 (G>T) point mutation.
 3. The method of claim 1, wherein thecell is homozygous for the c.4886+1249 (G>T) point mutation.
 4. Themethod of claim 2, wherein the cell contains a second DYSF gene encodinga wild-type DYSF protein.
 5. The method of claim 2, wherein the cellcontains a second DYSF gene comprising a mutation that causes apremature stop codon. 6.-10. (canceled)
 11. The method of claim 1,wherein the cell is a human cell. 12.-14. (canceled)
 15. A method ofmodulating splicing in a cell containing a DYSF gene that encodes apre-messenger RNA having a pseudoexon incorporated between exons 44 and45, the method comprising: delivering to the cell an antisense nucleicacid comprising 15 to 25 nucleotides in length that targets thepre-messenger RNA encoded by the human DYSF gene and alters splicing ofthe pre-messenger RNA such that the pseudoexon is not incorporatedbetween exons 44 and 45 of the pre-messenger RNA, wherein the antisensenucleic acid is complementary to a sequence as set forth in SEQ ID NO:56 or SEQ ID NO:
 58. 16.-20. (canceled)
 21. The method of claim 1,wherein the antisense nucleic acid is expressed from a transgene. 22.The method of claim 21, wherein the transgene is delivered to the cellusing a viral vector.
 23. The method of claim 22, wherein the viralvector is a recombinant AAV vector. 24.-45. (canceled)
 46. The method ofclaim 1, wherein the antisense nucleic acid comprises at least onemodified nucleotide.
 47. The method of claim 46, wherein the at leastone modified nucleotide is a 2′-modified nucleotide, optionally whereinthe 2′-modified nucleotide is selected from the group consisting of2′-deoxy, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, 2′-amino and2′-aminoalkoxy modified nucleotides.
 48. The method of claim 47, whereinthe 2′-modified nucleotide is selected from the group consisting of2′-deoxy, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, 2′-amino and2′-aminoalkoxy modified nucleotides.
 49. (canceled)
 50. The method ofclaim 1, wherein the antisense nucleic acid comprises at least onemodified internucleotide linkage.
 51. The method of claim 50, whereinthe at least one modified internucleotide linkage is a phosphorothioatemodified linkage. 52.-78. (canceled)
 79. An expression constructencoding an antisense nucleic acid having a region of complementaritythat is complementary with a sequence between exons 43 and 46 encoded bya human DYSF gene.
 80. The expression construct of claim 79, wherein thehuman DYSF gene comprises a c.4886+1249 (G>T) mutation.
 81. (canceled)82. The expression construct of claim 79, wherein the region ofcomplementarity is complementary with at least 8 contiguous nucleotidesof a sequence as set forth as SEQ ID NO: 116, 117, 118, 119, 120 or 121.83.-86. (canceled)
 87. The expression construct of claim 79, wherein theantisense nucleic acid expressed from the vector, when present in a cellthat contains the human DYSF gene, alters splicing of a pre-messengerRNA expressed from the human DYSF gene such that the pseudoexon is notincorporated between exons 44 and
 45. 88. A recombinant AAV comprisingthe expression construct of claim
 79. 89.-110. (canceled)